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Genome-Editing-Mediated Restructuring of Tumor Immune Microenvironment for Prevention of Metastasis

  • Dongyoon Kim
    Dongyoon Kim
    College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
    More by Dongyoon Kim
  • Yina Wu
    Yina Wu
    College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
    More by Yina Wu
  • Gayong Shim*
    Gayong Shim
    School of Systems Biomedical Science, Soongsil University, Seoul 06978, Republic of Korea
    *E-mail address: [email protected]
    More by Gayong Shim
  • , and 
  • Yu-Kyoung Oh*
    Yu-Kyoung Oh
    College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
    *E-mail address: [email protected]. Tel: 82-2-880-2493. Fax: 82-2-882-2493.
    More by Yu-Kyoung Oh
Cite this: ACS Nano 2021, 15, 11, 17635–17656
Publication Date (Web):November 1, 2021
https://doi.org/10.1021/acsnano.1c05420

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

Modulating the tumor immune microenvironment to activate immune cells has been investigated to convert cold to hot tumors. Here, we report that metal–lipid hybrid nanoparticle (MLN)-mediated gene editing of transforming growth factor-β (TGF-β) can restructure the tumor microenvironment to an “immune activated” state for subsequent immunotherapy. MLNs with cationic lipids and elemental metallic Au inside were designed to deliver plasmid DNA encoding TGF-β single guide RNA and Cas9 protein (pC9sTgf) and to convert near-infrared light (NIR) to heat. Upon NIR irradiation, MLNs induced photothermal anticancer effects and calreticulin exposure on B16F10 cancer cells. Lipoplexes of pC9sTgf and MLN (pC9sTgf@MLN) provided gene editing of B16F10 cells and in vivo tumor tissues. In mice treated with pC9sTgf@MLNs and NIR irradiation, the tumor microenvironment showed increases in mature dendritic cells, cytotoxic T cells, and interferon-γ expression. In B16F10 tumor-bearing mice, intratumoral injection of pC9sTgf@MLNs and NIR irradiation resulted in ablation of primary tumors. Application of pC9sTgf@MLNs and NIR irradiation prevented the growth of secondarily challenged B16F10 cells at distant sites and B16F10 lung metastasis. Combined TGF-β gene editing and phototherapy is herein supported as a modality for restructuring the tumor immune microenvironment and preventing tumor recurrence.

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Recently, tumor immune microenvironments have gained attention for immunotherapy. Tumor immune microenvironments are critical factors in effective immunotherapeutic outcomes. The heterogeneity of the tumor microenvironment and the complex interactions between tumor cells and immune cells can hamper clinical outcomes in patients. (1−3) Cold tumors exhibit limited infiltration of immune cells, whereas hot tumors show increased infiltration of macrophages and T cells. For immunotherapy, it could be critically helpful to restructure the tumor immune microenvironment to increase immune cell infiltration. (4)
Modulation of tumor immune microenvironments has been studied using various nanomaterials. (5−9) For example, lipid/polymer hybrid nanomaterial-mediated nitric oxide delivery was reported to normalize tumor vessels and convert an immunosuppressive cold tumor to an immunostimulatory hot tumor in hepatocellular carcinoma. (6) A pH- and reactive oxygen species-responsive nanogel was investigated for delivery of anti-programmed cell death protein 1 antibody to reverse the immunosuppressive tumor microenvironment. (7) Co-delivery of oxaliplatin and indoleamine 2,3-dioxygenase 1 inhibitor was designed to induce immunogenic cell death and modulate the tumor immune microenvironment. (8) Very recently, collagen-binding domain fused to interleukin-12 was shown to change the immune microenvironment of tumors and increase the intratumoral level of interferon-γ (IFN-γ). (9)
Although progress has been made in this field, few studies have sought to modulate the tumor immune microenvironment by gene editing of key cytokines known to contribute to immune evasion. Such cytokines include transforming growth factor (TGF)-β, which has been shown to be involved in the immune evasion of the tumor microenvironment. (10−12) TGF-β widely interacts with various immune cells, such as neutrophils, natural killer cells, dendritic cells, macrophages, regulatory T (Treg) cells, Th1 helper cells, and cytotoxic T cells, in the tumor microenvironment. Through interactions with immune cells, TGF-β can induce a tumor immune microenvironment that is favorable for immune evasion. (10) TGF-β can form an immunosuppressive tumor microenvironment by promoting the differentiation of Treg cells and interrupting the generation of the Th1-effector phenotype. (10,11)
In this study, we hypothesized that gene editing of TGF-β could restructure the tumor immune microenvironment with activated immune cells. To test this hypothesis, we constructed a plasmid DNA encoding clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) and single guide RNA (sgRNA) specific for TGF-β (pC9sTgf) and delivered pC9sTgf in complexes with cationic metal and lipid hybrid nanoparticles (MLNs) (Figure 1). In MLNs, Au metal was encapsulated to enable a photothermal effect, whereby the surface exposure of damage-associated molecular patterns (DAMP) on tumor cells is increased upon near-infrared (NIR) irradiation. Here, we report that the TGF-β gene editing in B16F10 tumor-bearing mice with the lipoplexes of pC9sTgf and MLNs (pC9sTgf@MLNs) followed by NIR irradiation could restructure the tumor immune microenvironment to have greater populations of infiltrated T cells, ablate primary tumors, and prevent distant secondary tumor formation and metastasis.

Figure 1

Figure 1. Construction of pC9sTgf@MLNs and proposed mechanisms for its restructuring of the tumor immune microenvironment. (A) Cationic MLNs containing Au metal clusters were prepared by reducing Au3+ ion with ascorbic acid. For Cas9/sgRNA-mediated gene editing of TGF-β, pC9sTgf was complexed to MLNs to form pC9sTgf@MLN lipoplexes. (B) TGF-β contributes to the immune suppressive tumor microenvironment through diverse signaling on various immune cells. pC9sTgf@MLN-mediated TGF-β gene editing restructures the tumor immune microenvironment to an “immune-activated” state. The phototherapy-induced exposure of the “eat-me” signal will enable tumor antigen uptake by activated dendritic cell and induce the tumor antigen-specific immune memory system.

Results and Discussion

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Characteristics of Nanoparticles

MLNs were characterized for their morphology, constituent element, properties, and particle size, as depicted in Figure 2A. As illustrated in Figure S1A, elemental Au was entrapped in lipid nanoparticles by reducing Au3+ with ascorbic acid-containing buffer. The structures of various plasmid DNA and MLN complexes are illustrated in Figure S1B. Inside MLNs, reduced and agglomerated Au clumps were observed (Figure 2B). Transmission electron microscopy (TEM) images revealed that MLNs had a characteristic multirounded particle shape and were filled with gold metal (Figure 2B), whereas lipid nanoparticles (LNs) showed the smooth particle shape of liposomes (Figure S2A,B). Elemental analysis showed that Au and P were present in MLNs (Figure 2C), whereas only P was present in LNs (Figure S2C). In the absence of plasmid DNA complexation, both LNs and MLNs were similar in their phosphorus contents; however they differed in Au contents. The concentrations of phosphorus in LNs and MLNs were 17.8 ± 1.2 μg/mL and 16.4 ± 1.1 μg/mL, respectively (Figure 2D). The concentration of Au was 1.3 ± 0.1 μg/mL in MLNs, whereas it was negligible in LNs.

Figure 2

Figure 2. Physicochemical features of MLNs. (A) Schematic illustration of MLNs with reduced elemental Au inside. (B) TEM image of MLNs. Scale bar = 50 nm. (C) Elemental composition of MLNs was analyzed by high-angle annular dark-field (HAADF) microscopy and energy-dispersive X-ray spectroscopy-scanning TEM (STEM-EDS). Elemental Au and P are shown in green and red colors, respectively. Scale bar = 50 nm. (D) Contents of elemental Au and P in nanoparticles measured by phosphate assay and inductively coupled plasma-mass spectrometry (ICP-MS), respectively (n = 5 per group). (E) Light absorbance spectra of nanoparticles measured from 300 to 1000 nm. (F) Colors and thermal images after NIR irradiation for LNs and MLNs. (G) Temperature of samples monitored during three times of repeated cycles of 808 nm NIR laser irradiation at a power of 1.5 W/cm2 with 4 min pulse and 4 min pause. (H) Gel retardation of pDNA@MLNs complexed at various N/P weight ratios observed on a 1% agarose gel. (I) Size distribution of MLNs or pDNA@MLNs measured by dynamic light scattering.

Unlike LNs, MLNs showed a broad light absorbance over 600–900 nm (Figure 2E) and were gray in color (Figure 2F). LNs and MLNs differed in their NIR-responsiveness. LNs did not show any temperature change upon NIR irradiation. In contrast, MLNs showed NIR-responsive temperature cycles, increasing to 51.5 ± 0.8 °C (Figure 2F,G) when the laser was on and decreasing to 28.7 ± 0.2 °C when the laser was off (Figure 2G). Our gel retardation assay showed that lipoplexes were formed between plasmid DNA and LNs (Figure S2D) or MLNs (Figure 2H). In MLNs, complete gel retardation of green fluorescent protein (GFP) expressing plasmid (pGFP) was observed at a N/P ratio (molar ratio of nitrogen in positive amine groups of nanoparticles to phosphorus in negative phosphate groups of plasmid DNA) of 7:1. The zeta potential of pGFP-complexed MLNs (pGFP@MLNs) increased with the N/P ratio (Figure S3). Based on the gel retardation results, the N/P ratio of 7:1 was used in this study. The mean particle sizes of MLNs and pGFP@MLNs were 93.5 ± 21.5 nm and 113.4 ± 18.8 nm, respectively (Figure 2I). Both MLNs and pGFP@MLNs remained stable in physiological conditions for 7 days (Figure S4).

Intracellular Fate

The efficiency of MLNs in delivering plasmid DNA was evaluated by monitoring the endosomal escape of the Cas9-expressing plasmid pSpCas9(BB). B16F10 cells were treated with lipoplexes of fluorescent dye-labeled DNA and MLNs with or without the fusogenic lipid 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), and endolysosome labeling was performed by LysoTracker staining. To evaluate the endosomal escape of the plasmid, signal colocalization was observed over 24 h (Figure 3A, Figure S5). Negligible endosomal escape was observed in cells treated with MLNs lacking DPhPE (Figure S5), whereas cytoplasmic diffusion of DNA from endolysosomes was observed from 4 h after the cells were treated with lipoplexes comprising MLNs with DPhPE (Figure 3A).

Figure 3

Figure 3. Cellular uptake and intracellular fate of plasmid DNA and nanoparticles. (A) B16F10 cells were treated with lipoplexes of red dye labeled DNA with MLNs for various durations and stained with LysoTracker (green color). The intracellular fate of DNA was monitored by confocal microscopy for 24 h. Scale bar = 10 μm. (B) Fluorescence signal of GFP was observed under confocal microscopy. Scale bar = 250 μm. (C) Transfected cells were analyzed by flow cytometry (n = 3 per group). (D) pGFP@MLN-treated cells were visualized by dark-field microscopy. Scale bar = 250 μm. (E) Au content of the cells was measured by ICP-MS (n = 3 per group). (F) Endocytosis of pGFP@MLNs was observed in B16F10 cells by TEM imaging (n.s., not significant; ***p < 0.001).

The transfection efficiency of MLNs was evaluated by expression of GFP encoded in pGFP. For comparison with pGFP@MLNs, lipoplexes formed with Lipofectamine 2000 (pGFP@LF) were used. Transfection of B16F10 cells with various lipoplexes of pGFP yielded comparable expression of GFP fluorescence among commercial transfection agent, LNs, and MLNs (Figure 3B). Flow cytometry showed that there was no significant difference in GFP-positive cell populations among LF, LNs, and MLNs (Figure 3C). The GFP-positive populations in cells treated with pGFP@LNs and pGFP@MLNs were 31.2 ± 2.1% and 31.1 ± 2.8%, respectively. The dark-field microscopy images of pGFP@LF and pGFP@LNs were comparable, whereas those of pGFP@MLNs showed bright spots due to Au clusters (Figure 3D). ICP-MS showed the presence of Au only in the cells treated with pGFP@MLNs, at a concentration of 13.4 ± 0.1 μg/L (Figure 3E). TEM images confirmed the uptake of MLNs by revealing dark spots representing the clustering of Au in the cells (Figure 3F).

Photothermal Effect and Calreticulin Exposure of pGFP@MLN-Treated Cells

The lipoplexes of pGFP@MLNs provided photothermal responsiveness and induced the exposure of calreticulin upon NIR irradiation. Treatment of B16F10 cells with pGFP@LNs did not yield any significant temperature change upon NIR irradiation (Figure 4A). pGFP@MLNs, in contrast, provided an increase of temperature from 27.0 °C to 52.9 ± 3.8 °C upon NIR irradiation (Figure 4B). No significant temperature change (Figure S6A) or cell death (Figure S6B) was observed in the pGFP@MLN-treated groups without NIR irradiation.

Figure 4

Figure 4. NIR responsiveness and exposure of calreticulin. The temperatures of B16F10 cells transfected with various nanoparticles were monitored by thermal imaging during NIR irradiation. The highest temperature reached by each group was visualized (A) and plotted (n = 3 per group) (B). One day after this treatment, the viability of B16F10 cells was visualized by live (green) and dead (red) cell staining (C) and by MTT assay (D) (n = 5 per group). (E) Cellular apoptosis was detected by annexin V and PI staining (n = 5 per group). (F) The exposure of calreticulin was analyzed by flow cytometry. The calreticulin-positive cell populations (G) and mean fluorescence intensity (MFI) (H) were quantified (n = 3 per group; ***p < 0.001).

pGFP@MLN-treated and NIR-irradiated (pGFP@MLN(+)) B16F10 cells showed a significant decrease in viability (Figure 4C,D), exhibiting 17.0-fold lower viability compared to the pDNA@LN(+)-treated group (Figure 4D). The apoptotic cell population, positive for annexin V and propidium iodide (PI), was significantly increased in cells treated with pGFP@MLNs plus NIR irradiation: the apoptotic population was 7.3 ± 0.4% in the pGFP@LN-treated and NIR-irradiated group (pGFP@LN(+)) (Figure 4E, Figure S7) but 93.8 ± 2.4% in the pGFP@MLN-treated and NIR-irradiated group (pGFP@MLN(+)).
The calreticulin-positive cell population was less than 10% in the untreated group and those treated with pGFP@LN(+). However, pGFP@MLN(+) treatment yielded notable up-regulation of calreticulin on the cell surface, with a calreticulin-positive cell population higher than 90% (Figure 4F,G). The mean fluorescence intensity (MFI) of the pGFP@MLN(+) treated group was 24.1-fold higher than that of the pGFP@LN(+) treated group (Figure 4H). The up-regulation of calreticulin exposure significantly enhanced the phagocytosis of cancer cells by dendritic cells, which was 2.3-fold higher for pGFP@MLN(+)-treated B16F10 cells than pGFP@LN(+)-treated B16F10 cells (Figure S8).

Genome-Editing Efficacy of MLNs in GFP-Expressing Cells and Tumor-Bearing Mice

To test the genome-editing efficacy of MLNs in GFP-expressing cells, various plasmid DNAs were constructed as illustrated in Figure S1B. Delivery of plasmid DNA encoding Cas9 and sgRNA specific for GFP (pC9sGfp) in MLN complexes provided genome-editing effects in vitro and in vivo. GFP-expressing cancer cells were treated with pC9sGfp complexed to MLNs (pC9sGfp@MLNs) (Figure 5A). After transfection, the efficiency of genome editing was evaluated by GFP measurement of fluorescence. Transfection of GFP-expressing cells with Cas9-encoding plasmid DNA-complexed MLNs (pC9@MLNs) or with plasmid DNA encoding Cas9 and scrambled sgRNA-complexed MLNs (pC9sScr@MLNs) did not significantly affect the fluorescence intensity of the cells (Figure 5B).

Figure 5

Figure 5. Gene-editing ability of pC9sGfp@MLNs. (A) To visualize the gene-editing ability, pC9sGfp was delivered by MLNs to GFP-stable cells. (B) Fluorescence signal of the HeLa-GFP cells was observed by confocal fluorescence microscopy after various transfections. (C) GFP-edited cells were analyzed by flow cytometry. (D) Efficiency of GFP gene editing was examined by T7 endonuclease 1 (T7E1) assay. A blue arrow indicates the amplified product of the target region, and red arrows indicate the cleavage products. (E) HeLa-GFP-xenografted mice received pC9sGfp@MLNs twice at a 2-day interval. (F, G) One day after the last transfection, the fluorescence signals of tumors were observed by molecular imaging (n = 3 per group). (H) Tumors were sectioned for fluorescence microscopy (*p < 0.05; **p < 0.01).

The GFP gene-edited populations of cells treated with pC9@MLNs and pC9sScr@MLNs were 15.7 ± 1.6% and 17.0 ± 0.4%, respectively, which did not significantly differ from that of the untreated group 15.9 ± 0.2% (Figure 5C, Figure S9A). However, the transfection with pC9sGfp@MLNs significantly reduced the fluorescence intensity of the GFP-expressing cells, showing a GFP-edited population of 45.2 ± 9.8%. The efficiency of MLN lipoplexes in GFP gene editing was further confirmed by monitoring the insertion and deletion (indel) frequency (Figure 5D). No significant cleavage was detected in the pC9@MLN- or pC9sScr@MLN-treated groups. In contrast, the pC9sGfp@MLN-treated group showed distinct DNA cleavage, with an indel frequency of 35.8%. Next, we examined MLN-mediated gene editing in GFP-expressing tumor-bearing mice (Figure 5E). Intratumoral injection of pC9@MLNs or pC9sScr@MLNs was not associated with a significant change in the GFP fluorescence of tumors (Figure 5F, Figure S9B). In contrast, pC9sGfp@MLNs significantly reduced the GFP signal at the tumor site, showing a 65% reduction in fluorescence intensity compared to that seen in untreated mice (Figure 5F,G). Tumor tissue sections of pC9sGfp@MLN-treated mice also showed reductions in GFP-positive cells (Figure 5H, Figure S9C).

TGF-β Gene Editing and Modulation of the Tumor Immune Microenvironment

For modulation of the immune microenvironment, TGF-β was chosen as a target for gene editing (Figure 6A). pC9sTgf was constructed to coexpress Cas9 and TGF-β-specific sgRNA (Figure 6B, Figure S1B). Western blot analysis showed that transfection of B16F10 cells with pC9@MLNs, pC9sScr@MLNs, or pC9sTgf@MLNs yielded expression of Cas9 protein (Figure 6C). Genome sequencing of TGF-β exon 1 showed the occurrence of indels in samples treated with pC9sTgf@MLNs (Figure 6D). In groups treated with MLNs, pC9@MLNs, or pC9sScr@MLNs, no indel was observed. In contrast, the group treated with pC9sTgf@MLNs showed an indel percentage of 31.8% (Figure 6E), with no off-target effect (Figure S10). The secretion of TGF-β by B16F10 cells was not significantly different among the untreated, MLN-treated, and pC9sScr@MLN-treated groups. However, in line with the rate of indel occurrence, the secretion of TGF-β was 3.0-fold lower in the group treated with pC9sTgf@MLNs compared to the untreated group (Figure 6F).

Figure 6

Figure 6. MLN-mediated gene editing effect on secretory TGF-β. (A) TGF-β-edited B16F10 cells are expected to exhibit reduced secretion of TGF-β. (B) Sequence structure of pC9sTgf and the sgRNA binding position in exon 1 of the TGF-β gene. (C) Expression of Cas9 protein in nanoparticle-treated cells was confirmed by Western blot analysis. (D) Representative sequencing analysis of polymerase chain reaction (PCR) amplicons of target region. A red arrow indicates a cleavage site and dotted lines indicate the deleted region. (E) TGF-β editing efficiency was observed by T7E1 endonuclease assay. (F) Concentration of secretory TGF-β in pC9sTgf@MLN-treated B16F10 cells was measured by ELISA (n = 5 per group; ***p < 0.001).

To evaluate the effect of TGF-β gene editing on immune cells, splenic T cells were treated with the media from cultures of cells treated with various lipoplexes. The populations of Treg cells and IFN-γ+ CD8+ cells were analyzed (Figure 7A, Figure S11). The group treated with pC9sTgf@MLNs showed the lowest population of FoxP3+ Treg cells (Figure 7B) and the highest population of IFN-γ+ CD8+ cells (Figure 7C). The population of Treg cells in the pC9sTgf@MLN-treated group was 1.6-fold lower than that in the group treated with pC9sScr@MLNs (Figure 7D). The population of IFN-γ+ CD8+ cells in the group treated with pC9sTgf@MLNs was 5.3-fold higher than that in the group treated with pC9sScr@MLNs (Figure 7E).

Figure 7

Figure 7. T cell differentiation by MLN-mediated TGF-β editing. (A) B16F10 cells were treated with various groups of nanoparticles, and the supernatants were applied to splenocytes. Two days later, the populations of Treg cells and IFN-γ+ CD8+ cells were analyzed. (B) Populations of FoxP3+ Treg cells were tested by flow cytometry. (C) Populations of IFN-γ+ CD8+ cells were tested by flow cytometry. Critical gates were marked with boxes. (D, E) Quantified cell populations were plotted for Treg cells (D) and IFN-γ+ CD8+ cells (E) (n = 5 per group; *p < 0.05; ***p < 0.001).

TGF-β gene editing in cancer cells affected the expression of other inflammatory or immunosuppressive markers in the splenic T cells. The mRNA expression levels of the inflammatory markers perforin, granzyme B, and C-X-C motif chemokine ligand 10 (CXCL10) were 2.5-, 2.4-, and 5.4-fold higher, respectively, in the group treated with pC9sTgf@MLNs compared to the group treated with pC9sScr@MLNs (Figure S12). Meanwhile, the mRNA expression level of the immunosuppressive protein FoxP3 was 2.3-fold lower in the pC9sTgf@MLN-treated group compared to the pC9sScr@MLN-treated group. Finally, the mRNA expression level of TGF-β was 2.8-fold lower in cells treated with pC9sTgf@MLNs compared to those treated with pC9sScr@MLNs.

Antitumor Effects against Primary Tumors

To observe antitumor effects, mice were intratumorally injected three times with pC9sTgf@MLNs and irradiated with NIR after the last dose. This dosing regimen was designed based on our findings regarding the photothermal and calreticulin exposure triggering effects of pC9sTgf@MLN(+) (Figure S13). The interval between the last dose of pC9sTgf@MLNs and NIR irradiation was found to modulate the photothermal effect. NIR irradiation at 1 day after the last dose showed the highest photothermal effect, compared to that applied at 2 or 3 days after the last dose. Thus, we applied NIR irradiation at 1 day after the last dose for our experiments (Figure S13A). Regarding the dosing frequency of pC9sTgf@MLNs, we tested three dosing regimens of pC9sTgf@MLNs, as illustrated in Figure S13B. The application of three doses separated by 2-day intervals was found to provide a photothermal effect to 51.0 ± 1.6 °C (Figure S13B). The exposure of calreticulin increased with the temperature, showing the highest exposure at 50 °C (Figure S13C). We thus applied three doses at 2-day intervals in this study.
Intratumoral administration of pC9sTgf@MLNs and subsequent NIR irradiation provided greater antitumor effect against primary tumors. Upon NIR irradiation, the groups treated with MLNs alone or lipoplexes with MLNs showed temperature increases (Figure 8B) that were not significantly different (Figure 8C). However, these groups differed in their durable antitumor effects. In the groups treated with MLNs, pC9@MLNs, or pC9sScr@MLNs, NIR irradiation induced temporary decreases in tumor volume (Figure 8D). Unlike the other groups, treatment with pC9sTgf@MLNs and NIR irradiation yielded tumor ablation for over 27 days after tumor inoculation (Figure 8D). In the untreated groups, tumor growth was observed regardless of NIR irradiation (Figure S15A,B). In the untreated group without NIR irradiation, 0% survival was observed at day 25 after tumor inoculation (Figure 11C). In the untreated and NIR-irradiated group, 0% survival was observed at day 29 after tumor inoculation (Figure S15C).

Figure 8

Figure 8. Antitumor efficacy of MLN-mediated TGF-β editing. (A) B16F10-bearing C57BL6 mice were intratumorally injected with various lipoplexes in the three tumor immune microenvironments with a 2-day interval. One day after the third injection, tumor sites were irradiated with NIR. (B) Thermal images of NIR-irradiated mice were obtained using a thermal camera. (C) Temperatures of tumor sites with or without NIR irradiation were plotted (n = 5 per group; ns, not significant; ***p < 0.001). (D) Tumor volumes of various groups were monitored for over 27 days after tumor inoculation. (E) Appearance of mice treated in various groups was observed at 18 days after tumor inoculation.

Modulation of the Tumor Immune Microenvironment by TGF-β Gene Editing

TGF-β gene editing and subsequent NIR irradiation changed the levels of TGF-β protein and immune cell compositions of tumor immune microenvironment. Mice representing the tumor immune microenvironments were injected with pC9sTgf@MLNs and subjected to NIR irradiation, and tumor tissues were extracted for analysis of in vivo gene editing and immune cell populations (Figure 9A). In vivo editing of TGF-β gene in the tumor tissues was observed in the group treated with pC9sTgf@MLNs and NIR (pC9sTgf@MLN(+)) but not in the other groups (Figure 9B). Indel occurrence at the cleavage site was observed in the tumor tissues treated with pC9sTgf@MLNs (Figure 9C, Figure S18A). In sequences 1, 2, and 4 (Figure 9C), partial deletions of target gene were observed, while in sequence 3, random insertion was observed at the cleavage site. In contrast, immune cells such as T cells, dendritic cells, and macrophages did not show detectable indel frequency (Figure S18B,C). In tumor immune microenvironments, the population of cells secreting TGF-β was lowest in the group treated with pC9sTgf@MLN(+) and was 3.4-fold lower than that from untreated tumor (Figure 9D).

Figure 9

Figure 9. Activation of the tumor immune microenvironment by MLN-mediated TGF-β editing. (A) B16F10-bearing C57BL6 mice received three injections of pC9sTgf@MLNs at 2-day intervals. One day after the last transfection, tumor tissues were collected for analysis. (B) In vivo gene editing efficiency was measured by T7E1 endonuclease assay. (C) Representative indel sequences of target regions were analyzed by targeted deep sequencing. Red arrow indicates the cleavage site, and dotted lines indicate the deleted region. (D) Concentration of TGF-β was measured in the secretome obtained from tumor cells (n = 5 per group). (E) Populations of Treg cells, cytotoxic T cells, and mature dendritic cells in the tumor tissues were analyzed by flow cytometry. Critical gates were marked with boxes. Quantified cell populations were plotted for Treg cells (F), cytotoxic T cells (G), and mature dendritic cell (H) (n = 5 per group). (I) Concentration of IFN-γ was measured in the secretome from the tumor cells (n = 5 per group; ***p < 0.001).

Treatment of mice with pC9sTgf@MLN(+) affected the compositions of Treg cells, CD8+ T cells, and CD40+ dendritic cells in the tumor immune microenvironment (Figure 9E). Immune cell profiling revealed that pC9sTgf@MLN(+)-treated tumors had 2.9-fold fewer Treg cells (Figure 9F), 4.1-fold more CD8+ T cells (Figure 9G), and 6.9-fold more CD40+ dendritic cells (Figure 9H) compared to the immune cell populations of untreated tumor tissues. In addition, the level of IFN-γ in pC9sTgf@MLN(+) treated tumors was 39.4-fold higher than that of untreated tumors (Figure 9I).
Transcriptomic analysis was used to further evaluate the alteration of the tumor immune microenvironment by pC9sTgf@MLN(+). CD3+ cells in the pC9sTgf@MLN(+)-treated tumor immune microenvironment showed increased expression of cytotoxic T cell phenotype-related genes, including Gzma, Ifng, Gzmb, and Prf1 (Figure 10A). Meanwhile, the expression levels of Foxa1, Foxo3, and Rtkn2 were down-regulated in pC9sTgf@MLN(+) treated tumors.

Figure 10

Figure 10. Transcriptomic analysis and immune cell populations in the tumor microenvironment. (A) B16F10-bearing C57BL6 mice were treated with the various lipoplexes three times at 2-day intervals. One day after the last injection, tumor sites were irradiated by NIR. Tumor tissues were collected and RNA from CD3+ cells was used for next-generation sequencing transcriptomic analysis. (B) Tumor tissues were stained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclei (blue) and fluorescent dye-tagged anti-TGF-β antibodies (red). Scale bar = 2 mm. (C) Tumor tissues were stained with DAPI for nuclei (blue), fluorescent dye-tagged anti-TGF-β antibodies (red), fluorescent dye-tagged anti-CD8 antibodies (yellow), fluorescent dye-tagged anti-FoxP3 antibodies (sky blue), and fluorescent dye-tagged anit-CD31 antibodies (green). Scale bar = 50 μm.

Immunofluorescence staining of tumor sections was used to visualize how by pC9sTgf@MLN(+) altered the tumor immune microenvironment (Figure 10B). The distribution of TGF-β (red color) was decreased in tumor tissues treated with pC9sTgf@MLN(+). The population of FoxP3+ cells (sky blue color) was decreased in the pC9sTgf@MLN(+)-treated group compared to the other groups. In contrast, CD8+ (yellow color) cells were increased in pC9sTgf@MLN(+)-treated tumor tissues compared to the untreated and pC9sScr@MLN(+)-treated groups.

Anticancer Immune Responses against Distant Tumors

Treatment of mice with pC9sTgf@MLN(+) provided anticancer effects against secondarily challenged tumors. Mice were inoculated with primary B16F10 tumors, treated three times with the various lipoplexes, and NIR irradiated. On the same day that the NIR irradiation was applied, the mice were challenged with B16F10 cells on their opposite flanks (Figure 11A). Distant tumor growth was observed in groups treated with MLN(+), pC9@MLN(+), and pC9sScr(+) but not in the group treated with pC9sTgf@MLN(+) (Figure 11B). In the untreated group, all mice died by 25 days after primary tumor inoculation. In the groups treated with MLN(+), pC9@MLN(+), and pC9sScr(+), all mice died within 53 days after primary tumor inoculation. However, in the group treated with pC9sTgf@MLN(+), 100% survival was observed until 100 days after primary tumor inoculation (Figure 11C).

Figure 11

Figure 11. Antitumor effect of MLN-mediated TGF-β editing. (A) B16F10 tumor-bearing C57BL6 mice received pC9sTgf@MLNs or other lipoplexes three times at a 2-day interval. One day after the last transfection, tumors were irradiated with NIR, and mice were rechallenged with B16F10 tumor cells in the opposite flank. Volume of the rechallenged tumor (B) and survival of mice (C) were monitored. (D, E) Tumor draining lymph nodes were collected for analysis on day 13. Single-cell suspensions from the lymph nodes were stained with the dendritic cell maturation markers, CD40 (D) and CD86 (E), and analyzed by flow cytometry. Critical gates were marked with boxes. (F, G) Populations of CD11c+CD40+ cells (F) and CD11c+CD86+ cells (G) were quantified and plotted (n = 5 per group). (H) Populations of effector memory CD8+ T cells (CD3+CD8+CD44+CD62Llow) in distant tumor lymph nodes were quantified and plotted (n = 5 per group; ***p < 0.001).

To demonstrate the mechanism by which distant tumor growth was prevented by pC9sTgf@MLN(+), the maturations of dendritic cells and memory CD8+ T cells in tumor draining lymph nodes were investigated. Mice were treated with various lipoplexes three times and irradiated with NIR. One day after NIR irradiation, the lymph nodes were collected for immune cell analysis (Figure 11D,E). The populations of CD11c+ CD40+ dendritic cells (Figure 11D) and CD11c+ CD86+ dendritic cells (Figure 11E) were increased in the group treated with pC9sTgf@MLN(+). The population of CD11c+ CD40+ dendritic cells was 1.5-fold higher in the group treated with pC9sTgf@MLN(+) versus that in the group treated with pC9sScr@MLN(+) (Figure 11F). The population of CD11c+ CD86+ dendritic cells was 1.6-fold higher in the group treated with pC9sTgf@MLN(+) versus that in the group treated with pC9sScr@MLN(+) (Figure 11G).
In mice treated with pC9sTgf@MLN(+), we observed a significant increase in the populations of effector memory CD8+ T cells (CD3+ CD8+ CD44+ CD62Llow) in distant tumor draining lymph nodes (Figure 11H, Figure S19). The memory CD8+ T cell population in the group treated with pC9sTgf@MLN(+) was 3.0-fold higher than that in the group treated with pC9sScr@MLN(+).
The ability of pC9sTgf@MLN(+) to prevent distant tumor growth was observed for B16F10 challenge but not for challenge with other tumor cells, such as MC38 or EL4 cells. Mice treated with pC9sTgf@MLNs were irradiated with NIR. On that same day, the mice were inoculated with B16F10, MC38, or EL4 cells (Figure 12A). In B16F10-challenged tumors, no growth was observed. However, in the groups challenged with MC38 or EL4 cells, tumor growth was observed (Figure 12B). The treatment of primary B16F10 tumor-bearing mice with pC9sTgf@MLN(+) provided protection against metastasis. After primary B16F10 tumor-bearing mice were treated with pC9sTgf@MLNs and NIR-irradiated, B16F10 cells were intravenously challenged to induce lung metastasis (Figure 12D). Three weeks after the challenge injection, the lungs of B16F10-challenged but untreated mice showed several tumor nodules (Figure 12E), as visualized by hematoxylin and eosin staining. In contrast, the lungs of B16F10-challenged and pC9sTgf@MLN(+)-treated mice showed 11.7-fold fewer nodules compared to the untreated group (Figure 12F, Figure S21).

Figure 12

Figure 12. Antitumor efficacy against B16F10, MC38, and EL4 distant tumors and B16F10 lung metastasis. (A) B16F10-bearing C57BL6 mice were injected with pC9sTgf@MLNs three times at 2-day intervals. One day after the last injection, tumors were irradiated with NIR, and mice were rechallenged with B16F10, MC38, or EL4 tumor cells in the opposite flank. (B) Tumor volumes were monitored over 30 days after the primary B16F10 inoculation. (C) Appearance of mice was observed on day 27 after the primary tumor inoculation. (D) B16F10-bearing C57BL6 mice were treated with pC9sTgf@MLNs three times at 2-day intervals. One day after the last injection, tumors were irradiated with NIR and mice were intravenously rechallenged with B16F10. (E) Three weeks after the secondary B16F10 injection, lung tissues were sectioned and subjected to hematoxylin and eosin staining. (F) Numbers of B16F10 nodules in lungs were quantified (n = 5 per group; ns, not significant; ***p < 0.001).

In Vivo Safety of pC9sTgf@MLN(+)

Repeated treatments of mice with pC9sTgf@MLN(+) did not induce any change in the histology of organ sections, hematological parameters, and body weights. Hematoxylin and eosin staining revealed that both pC9sTgf@MLN- and pC9sTgf@MLN(+)-treated mice displayed normal histology in organs such as liver, heart, lung, spleen, and kidney (Figure S22). Biochemical parameters also supported no systemic liver or kidney failures. Blood samples of pC9sTgf@MLN- and pC9sTgf@MLN(+)-treated mice showed that the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) are in the normal range (Figure S23A). Regardless of treatments, all mice showed normal ranges of hematological parameters such as the numbers of red blood cells (RBCs), white blood cells (WBCs), neutrophils, lymphocytes, eosinophils, and basophils (Figure S23B). No significant body weight change was observed in the group treated with pC9sTgf@MLN(+) compared with the untreated group (Figure S24).

In Vivo Off-Target Effects and Autoimmune Responses

Intratumoral treatment of mice with pC9sTgf@MLNs did not trigger genome editing in nontumor tissues. Our indel analysis revealed that there was no disruption of the TGF-β gene in major organs, including the heart, lung, liver, kidney, and lymph nodes (Figure 13A). There was no difference between the pC9sTgf@MLN(+)-treated samples collected at day 13 versus day 30 after tumor inoculation. Further analysis revealed that the TGF-β levels in nontarget tissues did not show any significant change: the levels of TGF-β found in the organ secretomes did not significantly differ between pC9sTgf@MLN(+)-treated and untreated mice (Figure 13B).

Figure 13

Figure 13. Lack of off-target effects or autoimmune responses. B16F10 tumor-bearing mice were intratumorally injected with pC9sTgf@MLNs three times at 2-day intervals. In the pC9sTgf@MLN(+) group, the mice were irradiated with NIR 1 day after the last injection. (A) On days 13 and 30 after tumor inoculation, major organs and lymph nodes were extracted from mice. TGF-β gene editing was evaluated by T7E1 assay. (B) TGF-β levels were quantified by ELISA (n = 5 per group). (C) Serum levels of IL-6, CCL2, and CRP were analyzed by ELISA (n = 5 per group). (D, E) On days 13 and 30, blood samples were extracted from naive mice and pC9sTgf@MLN(+)-treated mice, and anti-nuclear antibody (ANA) (D) and anti-dsDNA antibody (E) were analyzed by ELISA (n = 5 per group; ns, not significant).

The treatment of mice with pC9sTgf@MLN(+) also did not induce cytokine release syndrome or the production of autoimmune antibodies. Cytokine release syndrome was evaluated by assessing for elevated serum levels of IL-6, C–C motif chemokine ligand 2 (CCL2), and C-reactive protein (CRP). However, no significant change was seen in the serum levels of IL-6, CCL-2, or CRP following treatment with pC9sTgf@MLN(+) (Figure 13C). Moreover, the serum levels of anti-nuclear antibody (ANA) and anti-double strand DNA (dsDNA) antibody did not show any significant difference between untreated and pC9sTgf@MLN(+)-treated mice (Figure 13D,E).
In this study, we developed MLNs, which are a multifunctional nonviral gene delivery system that enables simultaneous gene editing and photothermal therapy. By delivering CRISPR/Cas9-mediated TGF-β gene-editing tools, this system could decrease the tumor microenvironment level of TGF-β, which plays a central role in immune suppression. As a result, a favorable environment was created for immune cells to be active, and anticancer immunity was established. Our MLNs could further achieve combinational anticancer treatment by exhibiting a NIR-responsive photothermal effect that could effectively kill cancer cells. The anticancer immunity induced by tumor immune microenvironment modulation prevented tumor recurrence and metastasis, while the photothermal therapy killed the tumor.
Tumor immune microenvironment restructuring and photothermal therapy could potentiate each other. Photothermal therapy has emerged as a promising cancer treatment strategy due to its noninvasiveness and easy spatiotemporal control, but limited penetration of light through the tissue or insufficient distribution of heat to the center of the tumor can result in incomplete tumor ablation and the risk of tumor recurrence and metastasis. (13) In this sense, overcoming the immune-suppressive environment to enable immune cells to fight against the residual cancer cells could be an effective strategy for eradicating tumors. In addition, photothermal therapy could increase tumor-associated antigens and DAMP molecules, which could strengthen the anticancer immune response. (14)
We herein show that our MLNs effectively offer both gene editing and phototherapy. MLNs were complexed to gene-editing plasmid DNA, and notable target gene suppression by gene editing was demonstrated in a GFP expression model (Figure 5). The advantages of a nonviral gene editing system include benefits in terms of safety and payload. (15) Although viral methods are superior in delivery efficiency, they have suffered from limitations in the size of the inserted cargo gene and risks of mutagenesis and immunogenicity. (16) Lipid nanoparticle-based gene editing may have great potential to stabilize and deliver diverse plasmid DNA constructs to the nuclei of tumor cells.
In the present study, MLNs were composed of cationic 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC) and N′,N′-3β-[N-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), which can form a complex with plasmid DNA through electrostatic interaction. The lipoplex structure was shown to help the plasmid DNA resist enzymatic degradation (Figure S25). The enhanced stability of MLN-lipoplexed pC9sTgf compared to the naked form may be attributed to the tight electrostatic interaction between the plasmid and the cationic MLNs. Nucleases are known to cleave the phosphodiester bond of DNA. The interaction of negatively charged phosphate groups with cationic MLNs may hinder the recognition and digestion of DNA by nucleases.
In tumor tissues, pC9sTgf@MLN complexes located in the endosomes of tumor cells need to escape to the cytosol and traffic to the nucleus in order to be effective. The trafficking of plasmid DNA to nuclei was observed by assessing the colocalization of Cy5-labeled pC9sTgf with the cell nuclei of tumor tissues in vivo. Indeed, our results revealed that the nuclear trafficking of Cy5-labeled pC9sTgf gradually increased over the 24 h period following intratumoral dosing (Figure S26A,B).
To elucidate a potential mechanism through which DNA could be released from MLNs at the endosome, we tested the interaction of endosome-like vesicles with pC9sTgf@MLNs at pH 5.2. We found that pC9Tgf was released from MLNs upon incubation with endosome-like vesicles at this pH, which mimics the acidity of the endosomal environment (Figure S27A,B). Since the endosomal membrane is negatively charged, the interaction of MLNs with the endosome may neutralize the cationic charge of MLNs, resulting in dissociation of the plasmid DNA. The escape of plasmid DNA from the endosome may be attributed to the presence of DPhPE, a neutral lipid component of MLNs that has been reported to have fusogenic properties (Figure 3A, Figure S5). DPhPE can reportedly invert the phospholipid bilayer to the nonbilayer hexagonal phase and promote destabilization of the endosomal membrane. (17,18)
Here, the entrapment of elemental Au inside lipid nanoparticles enabled MLNs to be NIR-responsive, and we observed the upregulation of calreticulin in MLN-treated cells upon NIR irradiation (Figure 4). For preparation of MLNs, we first encapsulated ascorbic acid and allowed HAuCl4·3H2O to diffuse into the lipid bilayer. Ascorbic acid exists in a charged form at neutral pH and could not penetrate the hydrophobic lipid bilayer. Unlike ascorbic acid, HAuCl4·3H2O is neutral and known to diffuse into the lipid bilayer. A recent study reported diffusion kinetics of the gold ion complexes into liposomes using a three-dimensional simulation approach. (19) In the study, the diffusion of AuCl3·H2O or HAuCl4·3H2O but not ascorbic acid into the liposomes was found.
In this study, we observed that the MLNs showed rough surfaces (Figure 2B,C) and no distinct light absorbance at 520 nm (Figure 2E). Several studies reported that the morphology of gold-based nanomaterials could affect the light absorbance patterns. (20) Absorption around 520 nm has been reported to be typical for gold nanoparticles with smooth surfaces. (21) But, consistent with our findings, broad light absorbance without a distinct peak at 520 nm has been reported for gold nanoparticles with sea urchin-like rough surfaces. (22−24) In one study, gold nanoparticles with rough surfaces were found to show a broad absorbance spectrum over 500–1300 nm. (22) The lack of a distinct peak has been attributed in part to the red-shift and the plasmonic coupling effect among spiked structures protruding on the surfaces. (22)
We observed relatively dim spots in the dark field microscope picture in the group treated with pGFP-LNs (Figure 3D), but no signal in the quantified Au data (Figure 3E). The difference can be explained by the specificity of gold detection between the dark field microscopy used for Figure 3D and ICP-MS for Figure 3E. In dark field microscopy, specimens are generally visualized by detecting the scattered and diffracted light after passing through the objects. The dark field microscopy provided clear light scattering features in the presence of gold nanoparticles. Although the light scattering in the pGFP-LN-treated group can be increased due to the presence of nanoparticles in the sample, it does not specifically detect gold. Unlike the dark field microscopy, ICP-MS is specific for gold and showed the presence of gold only in the samples treated with pGFP-MLNs, but not with pGFP-LN.
As a representative gene-editing target, we chose TGF-β. It has been reported that the reduction of TGF-β levels might serve as a switch that can convert a “cold tumor” into a “hot tumor”. TGF-β appears to act as a key regulator of tumor immunity through its numerous roles in immunosuppression and oncogenesis. (25) TGF-β has been reported to be abundantly secreted from various cancer cell types and involved in diverse mechanisms of the tumor immune microenvironment. TGF-β is known to promote immune suppression by affecting various immune cells, including neutrophils, natural killer cells, dendritic cells, macrophages, Th1 helper cells, and cytotoxic T cells. (10,11) Given these crucial roles, gene editing against TGF-β could be an impactful anticancer strategy.
Our observations show that TGF-β gene editing in cancer could affect the Treg cell-mediated immune suppressive responses. It has been reported that TGF-β can support Treg cell differentiation and induce T cell tolerance. (11) Recently, the TGF-β receptor was knocked out in chimeric antigen receptor-T (CAR-T) cells to suppress Treg cell expansion and improve the therapeutic efficacy of CAR-T cells in solid tumor models. (26) In another study, gene editing of cytokines in CAR-T cells was investigated as a means to alleviate cytokine release syndrome. In the study, the gene encoding granulocyte–macrophage colony-stimulating factor, a key mediator of CAR-T cell cytokine secretion, was edited by transcription activator-like effector nucleases. (27)
We observed that TGF-β gene editing by pC9sTgf@MLNs decreased the Treg cell population (Figure 7) and the immune suppressive activity of the tumor microenvironment, as demonstrated by an increase of IFN-γ+ CD8+ T cells. The reduction of TGF-β by MLN-mediated gene editing decreased the Treg cells in tumor tissues. TGF-β signaling was reported to induce FoxP3 expression in CD4+ T cells by phosphorylation of Smad protein family members. (11) Inhibition of TGF-β signaling in CAR-T cells was shown to prevent the Treg cell conversion of CD4+ T cells. (26)
The TGF-β gene editing and phototherapy mediated restructuring of the tumor immune microenvironment provided complete tumor ablation and protection against tumor recurrence (Figure 8). Previously, combined treatment of anti-TGF-β antibody and anti-programmed death-ligand 1 antibody was shown to increase T cell infiltration in the tumor microenvironment and improve tumor response to immune checkpoint blockade. (28) The small molecule inhibitor of TGF-β receptor tyrosine kinase galunisertib (LY2157299) has shown antitumor efficacy against various malignancies and is currently in phase II trials for treatment of hepatocellular carcinoma (NCT01246986). However, in these studies, prevention of recurring tumors was not investigated. (29,30)
Analysis of dendritic cells in tumor draining lymph nodes indicated that TGF-β gene editing activated dendritic cells and the activated dendritic cells migrated to the lymph nodes to induce systemic anticancer immune protection (Figure 11). As a result, no tumor growth was observed upon secondary tumor challenge (Figure 9). Notably, this prevention of distant tumor growth was cancer cell-type dependent, indicating that the protection was derived from activation of an immune response in the tumor immune microenvironment (Figure 10). Our results also demonstrated that this strategy could effectively prevent metastasis once systemic immunity against the tumor was established (Figures 11 and 12).
Although we tested TGF-β as the gene editing target to restructure the tumor immune microenvironment, MLNs can be applied for gene editing of other cytokines or immune-modulating proteins. By modifying the sgRNA sequence to target various factors, the formulation can be used to adjust the tumor microenvironment in the desired direction. We herein demonstrate proof-of-concept for this system in B16F10 tumors. However, the combination of gene editing and photothermal therapy can be used to treat primary and secondary recurrence of other solid tumors.
In this study, we applied metal–lipoplex-mediated gene editing for modulation of the tumor immune microenvironment. Recent studies reported the combination of heat-triggering for enhancing nucleic acid delivery and for gene-editing efficiencies. (31−34) In one study, the sgRNA was hybridized with a protector DNA on the Au nanorod to facilitate the release of the sgRNA in response to a NIR-induced temperature increase. (32) A heat-inducible promoter was applied to the CRISPR/Cas9 expression system and a cationic polymer-coated Au nanorod was used to deliver gene editing, such that the gene editing was activated upon NIR irradiation. (33) A recent study reported that cationic peptide and lipid-coated Au nanoparticles could provide the heat-dependent release of CRISPR/Cas9 components and enhance editing of the polo-like kinase 1 gene for anticancer therapy. (34)
While the previous studies utilized photothermal treatment as a switch for controlling gene editing of oncogenes, the photothermal treatment of MLNs in our study was used as a local ablative therapeutic tool for the tumor with a restructured immune microenvironment. In this study, we hypothesized that the photothermal effect of pC9@MLNs may induce the exposure of DAMP signals on tumor cells. Indeed, we observed the enhanced exposure of calreticulin, one of the DAMP signals, on the light-irradiated tumor cells (Figure 4F). During in vivo study, the exposure of DAMP signal alone has limited effect in modulating the tumor immune microenvironment, and we observed the recurrence of tumors in the group treated with pC9@MLNs and NIR-irradiated (Figure 8D). The recurrence of tumors was inhibited only when the mice were treated with pC9sTgf @MLNs. The in vivo study supports the importance of DAMP signal exposure as well as the gene editing mediated modulation of immune environments.
Here, we tested the exposure of calreticulin as a representative DAMP signal upon NIR irradiation-mediated cell death or damage for cells treated with pGFP@MLNs. We observed the significant increase of NIR-responsive temperature and exposure of calreticulin in the group treated with pGFP@MLNs, but not in the group treated with pGFP@LNs (Figure 4B,F). These data support that the NIR-mediated photothermal effect of pGFP@MLNs is due to the existence of gold clusters in MLNs. The exposure of DAMP signals on the cell surfaces would increase due to various stresses and death conditions. (35,36) We observed that calreticulin-positive cell populations were higher than 90% in the group treated with pGFP@MLN(+). This population may include both heat-killed dead cells and damaged live cells with enhanced DAMP signals. We also observed that the treatment with pGFP@MLN(+) resulted in the death of more than 80% of cells (Figure 4D). Thus, the treatment with pGFP@MLNs and NIR irradiation is expected to increase the exposure of calreticulin by damaging cells. The exposure of calreticulin, an “eat me” signal, would then promote the phagocytosis of damaged or dead tumor cells by nearby antigen-presenting cells. Indeed, we observed that treatment with pGFP@MLN(+) significantly enhanced the phagocytosis of tumor cells by dendritic cells (Figure S8).
We observed in vivo MLN-mediated photothermal effects in all the groups treated with MLN(+), pC9@MLN(+), pC9sScr@MLN(+), and pC9sTgf@MLN(+) (Figure 8C). Notably, the groups treated with MLN(+), pC9@MLN(+), and pC9sScr@MLN(+) showed temporary suppression of tumor growth after NIR irradiation (Figure 8D) but regrowth of tumors over 27 days. Only the group treated with pC9sTgf@MLN(+) showed no regrowth of tumors after NIR irradiation (Figure 8D) and significantly higher maturation of dendritic cells (Figure 9H). The transient tumor suppression observed in the MLN(+), pC9@MLN(+), and pC9sScr@MLN(+) groups may be due to the heat-induced partial death of tumor cells and support the importance of additional gene editing of tumor cells. The NIR irradiation can induce the exposure of calreticulin on the cell surfaces, but the DAMP signal alone may not be sufficient to activate the immune cells in the tumor microenvironments. TGF-β is known to serve as a major factor contributing to the suppressive immune environment by directly regulating antitumor function of immune cells. The orchestrated reduction of TGF-β in the tumor microenvironment could be essential to potentiate the functions of immune cells. The treatment with MLN(+), pC9@MLN(+), or pC9sScr@MLN(+) did not control the levels of immune cells, showing significantly lower populations of CD3+CD8+ T cells (Figure 9G) and CD11c+ CD40+ dendritic cells (Figure 9H).
Consistent with our observation, several studies reported the importance of additional immune cell stimulations in tumor microenvironments. Combination of photothermal therapy with immune adjuvants has been reported to enhance the effects of cancer immunotherapy. (37) As immune adjuvants, CpG, imiquimod, and chitosan derivatives have been reported. (38−42) Immune checkpoint inhibition is another strategy to induce the photothermal-mediated immune activation. (43) Compared with those studies, this study employed gene editing of immunosuppressive cytokine TGF-β for immune modulation of tumor microenvironments.
The biodistribution kinetics of pC9sTgf@MLNs to various organs were traced using Au element analysis. Intratumorally injected pC9sTgf@MLNs showed the highest retention at the injection site over 8 days after the injection (Figure S28). Since MLNs were injected locally to the tumor tissue rather than being systemically administered, the amounts of Au detected in other major organs, including the liver, lung, heart, spleen, and kidney, were greater than 2 orders of magnitude lower than those in the tumor tissues. Four days after pC9sTgf@MLN injection, Au was detected in tumor tissues but undetectable in all of the sampled major organs. The intratumoral injection of pC9sTgf@MLNs did not induce any harmful alteration of tissue or blood. The histology of organs did not show any sign of abnormality. Hematological and biochemical parameter analyses showed that all parameters were in their normal ranges after intratumoral injection of pC9sTgf@MLNs (Figures S22 and S23).
When pC9sTgf@MLNs were injected into tumors, significant gene editing was observed only in tumor cells, not in dendritic cells, macrophages, or T cells (Figure S18). At the cellular level, pC9sTgf@MLNs showed higher uptake by tumor cells compared with various types of nontumor cells, including fibroblasts, adipocytes, dendritic cells, macrophages, and T cells. The uptake of pC9sTgf@MLNs was 4.7-fold and 247.7-fold higher in B16F10 cells than in fibroblasts and adipocytes, respectively. In addition, the uptake of pC9sTgf@MLNs was at least 13.8-fold higher in B16F10 cells than in dendritic cells, macrophages, and T cells (Figure S29A,B).
The viability of pC9sTgf@MLN-treated cells under NIR irradiation differed between tumor and normal cells (Figure S29D,E). NIR irradiation resulted in a significant temperature increase in B16F10 cells, but not in the other tested cells. Similarly, whereas the viability of pC9sTgf@MLN-treated B16F10 cells was 8.0 ± 1.7% after NIR irradiation, those of the other cells remained above 80%, regardless of NIR irradiation. The viabilities of NIR-irradiated normal cells were 80.2 ± 8.1% for fibroblasts and 93.2 ± 3.8% for adipocytes (Figure S29F). The viability of NIR-irradiated dendritic cells, macrophages, and splenic T cells was 82.5 ± 17.0%, 84.3 ± 12.5%, and 91.9 ± 4.5%, respectively (Figure S29F).
The exact mechanisms by which tumor cells exhibit higher uptake of pC9sTgf@MLNs than other types of cells will need to be investigated further. However, we speculate that surface membrane charges between tumor and normal cells may contribute to this difference. It has been reported that cancer cells tend to have greater negative surface charges due to their higher membrane exposure of negatively charged phosphatidyl serine and overexpression of negatively charged sialic acid. (44,45) In one study, fluorescent dye-conjugated cationic nanoparticles were found to exhibit increased distribution to tumor cells due to electrostatic interaction. (46) In the present study, we measured the zeta potentials of the cancer cells and other tested normal cells and found that the average zeta potential of the cancer cells was −30.3 ± 4.6 mV, whereas those of the other cells were less than −21.68 mV (Figure S29C).
TGF-β is a major immunosuppressive cytokine that is known to drive the immune evasion of cancer. TGF-β has been reported to promote tumor metastasis through the epithelial–mesenchymal transition. (47) In cancer patients, an elevated level of TGF-β is a significant indicator of poor prognosis. For anticancer therapy, various TGF-β inhibitors, such as neutralizing antibodies, antisense oligonucleotides, and small chemicals, have been investigated. (48,49) Contrary to the initial expectations, TGF-β blockade alone has not been successful in clinical trials and has not yet been approved as a therapeutic option. (50)
One of the major therapeutic limitations of TGF-β blockade is that it lacks the ability to directly kill or suppress cancer cells. It seems unlikely that a therapeutic effect will be achieved solely through the inhibition of signaling pathways. Therefore, it is critical to select appropriate therapies that may be combined with TGF-β blockade to enable successful treatment. Another clinical consideration is the duration of TGF-β blockade. Repeated administration of TGF-β inhibitors is required to maintain an effective concentration, but it can induce resistance, leading to poor therapeutic outcomes. Moreover, systemic side effects are considered major clinical challenges, since TGF-β contributes to diverse physiological functions. In clinical trials, significant cardiotoxicity and cytokine release syndrome have been reported after systemic administration of TGF-β inhibitors. (51,52)
In the present study, we aimed to overcome the limitation of conventional TGF-β blockade strategies that use chemical drugs or antibodies by designing a nanoformulation that can prolong the blocking effect of TGF-β via genome editing. When administered by intratumoral injection, we expected that our nanoformulation would yield a localized genome editing effect in the tumor microenvironment. Indeed, intratumorally administered pC9sTgf@MLNs showed temporary retention to tumor tissues and significantly lower distribution to other organs, such as the liver (Figure S28). We observed no evidence of an autoimmune response or cytokine release syndrome (Figure 13). In addition, we combined this gene editing strategy with NIR irradiation, which, due to the presence of reduced gold in pC9sTgf@MLNs, increased the temperature of nanoparticle-bearing tumor cells and enhanced their phagocytosis and processing by nearby dendritic cells.

Conclusion

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In conclusion, this study supports the potential of gene editing to change the tumor immune microenvironment to ignite systemic immune responses and protect against secondary tumor challenge. The presence of elemental gold and the cationic charges of MLNs enabled Cas9 gene editing and photoimmunotherapy to be undertaken in a single carrier. Although we studied the effect of gene-edited tumor microenvironment using an sgRNA against TGF-β, the MLN nano-delivery system of Cas9 plasmid and sgRNA can be broadly extended to modulate other target genes.

Experimental Section

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Cas9 Plasmid DNA Construction for Gene Editing

The sgRNA sequences for target genes were designed using the Wellcome Trust Sanger Institute online platform (http://www.sanger.ac.uk/htgt/wge/). Plasmid DNA for gene editing was prepared by cloning the oligo duplexes for the sgRNA into a Cas9-expressing plasmid (pSpCas9(BB); Addgene, Watertown, MA, USA) according to the previously reported protocol. (53) The sequences of the oligomers for transcribing the sgRNA targeting GFP or TGF-β are provided in Supplementary Table S1. Briefly, 100 μmol of oligo duplexes was phosphorylated by T4 polynucleotide kinase (New England Biolabs (NEB), Ipswich, MA, USA) and annealed. The resulting hybridized oligo duplexes were inserted into pSpCas9(BB) plasmid predigested with BbsI restriction enzyme (Thermo-Fisher Scientific, Waltham, MA, USA). After 1 h, the residual linearized DNA was removed by digestion with exonuclease (Lucigen, Middleton, WI, USA). The ligated plasmid was transformed into DH5α competent cells (ECOS 101; Yeastern Biotech, Taipei, Taiwan) by heat shock method at 42 °C for 100 s, and the cells were plated on an LB agar plate containing 100 μg/mL of ampicillin (Sigma-Aldrich, Saint Louis, MO, USA). The plasmid was extracted with a PureLink HiPure Plasmid Midiprep Kit (Thermo-Fisher Scientific) according to the manufacturer’s instruction, and sgRNA oligo duplex insertion was confirmed by Sanger sequencing with the U6-Fwd primer (5′-GAGGGCCTATTTCCCATGATTCC-3′) (Macrogen Inc., Daejeon, Republic of Korea).

Evaluation of Gene Editing in Genomic DNA

The occurrence of gene editing was tested using T7E1 endonuclease, which recognizes mutations due to editing. The specific region of genomic DNA that covered the sgRNA target sequence was amplified by PCR using the primer sequences listed in Supplementary Table S1. The resulting PCR products were purified using a QIAquick Gel extraction kit (Qiagen, Hilden, Germany), and 200 ng of the purified PCR products was diluted with NEBuffer 2 (NEB). Heteroduplexes were formed under the following conditions: denaturation at 90 °C for 5 min, annealing at 95–85 °C (rate = −2 °C/s) and 85–25 °C (rate = −0.1 °C/s). The resulting heteroduplexes were incubated with 1 μL of T7E1 endonuclease and electrophoresed on a 2% agarose gel. The percentage of insertion and deletion (indel) was calculated with the ImageJ software (NIH, Bethesda, MD, USA) according to the formula 100 × (1 – [1 – (b + c)/(a + b + c)]1/2), where a is the integrated intensity of the uncleaved PCR product and b and c are the integrated intensities of each cleaved fragment. (53) A representative indel sequence in the genomic cleavage site was analyzed by Sanger sequencing (Macrogen Inc.).

Preparation of Lipid Nanoparticles

Various lipid nanoparticles were prepared by thin-film hydration and gold clustering methods. (19,54) For gold clustering, ascorbic acid (Sigma-Aldrich) was used as a reducing agent. Briefly, 0.622 μmol of EDOPC (Avanti Polar Lipids, Alabaster, AL, USA), 0.232 μmol of DC-Chol (Avanti Polar Lipids), and 0.146 μmol of DPhPE (Avanti Polar Lipids) were dissolved in chloroform. For the cellular uptake test, 0.02 μmol of Cy5-conjugated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:1 Cy5 PE; Avanti Polar Lipids) was added as a component of the lipid nanoparticles. The organic solvent was removed under vacuum to form lipid thin films. For preparation of plain lipid nanoparticles (LNs) as a control, the resulting lipid thin films were hydrated with 0.5 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 20 mM, pH 7.0). For the preparation of MLNs, the lipid thin films were hydrated with 0.5 mL of 20 mM HEPES buffer containing ascorbic acid (49 mM) and sonicated. The resulting ascorbic acid-loaded nanoparticles were purified from free ascorbic acid using a PD spin column (GE Healthcare, Buckinghamshire, UK), mixed with 25 nmol of 10 mM HAuCl4 solution (Sigma-Aldrich), and incubated for 10 min. (19) The obtained MLNs were purified from free Au3+ ions through membrane filtration (cutoff 100 kDa; Merck Millipore, Burlington, MA, USA) and stored at 4 °C until use.

Characterization of Nanoparticles

Nanoparticles were characterized by size, zeta potential, morphology, and elemental analysis. The mean size and distribution of nanoparticles were measured by dynamic light scattering technique using an ELSZ-1000 (Otsuka Electronics Co., Osaka, Japan). The morphology was observed using a TEM (Talos L120C; Thermo-Fisher Scientific). For analysis of element composition, elemental mapping was conducted using an STEM-EDS (JEM-2100F; JEOL, Tokyo, Japan).
Elemental Au was quantified by ICP-MS (Varian 820-MS; Varian, Palo Alto, CA, USA). Elemental phosphorus was quantified by phosphate assay using a phosphorus standard solution (Sigma-Aldrich). (54) Photoresponsive cycles of MLNs were monitored by thermal imaging. LNs or MLNs were irradiated with an 808 nm NIR diode laser (BWT Beijing LTD, Beijing, China) at a power of 1.5 W for 4 min and then cooled for 4 min; the cycle was repeated three times. NIR-responsive changes of temperature were recorded using a FLIR T420 IR thermal imaging system (FLIR System Inc., Danderyd, Sweden).
The complexation of plasmid DNA with MLNs was evaluated by a gel retardation assay. (55) Briefly, 100 ng of plasmid DNA encoding GFP (pGFP, cat. no. 6085-1; Clontech, Palo Alto, CA, USA) was mixed with MLNs at various nitrogen/phosphorus (N/P) weight ratios and incubated for 10 min. The resulting pGFP@MLN lipoplexes were electrophoresed on a 1% agarose gel (Mupid-2plus; Takara Bio Inc., Kusatsu, Japan). To test the stability in a physiological environment, the nanoparticles were stored at 4 °C in 20 mM phosphate-buffered saline supplemented with 10% fetal bovine serum. The sizes of nanoparticles were measured by dynamic light scattering over 7 days.

Zeta Potential Measurement

The zeta potentials of nanoparticles were measured by laser Doppler microelectrophoresis. Nanoparticles were dispersed in 20 mM HEPES buffer (pH 7.0) and measured using an ELSZ-1000 (Otsuka Electronics Co.). In some experiments, the indicated cells were suspended in 20 mM phosphate buffered saline, and their zeta potentials were measured.

Cellular Uptake and Intracellular Distribution Study

Cellular uptake of pGFP@MLN lipoplexes was measured by analyzing the amounts of elemental Au. B16F10 melanoma cells (Korean Cell Line Bank, Seoul, Republic of Korea) were seeded in a 24-well plate at a density of 1 × 105 cells per well and incubated overnight. The cells were then treated with pGFP@MLNs formed at a N/P weight ratio of 7:1. After 12 h, the medium was replaced, and the cells were incubated for 2 days. Nanoparticles in the cells were qualitatively observed using dark field microscopy (Axioimager M1; Carl Zeiss). For quantitative analysis of cellular uptake, the cell samples were digested with 90% nitric acid for 2 days and analyzed by ICP-MS (Varian 820-MS; Varian).
The intracellular distribution of MLNs in cancer cells was observed by TEM. Transfected cells were fixed with Karnovsky’s solution. (41) The pellets were washed with 0.05 M sodium cacodylate, fixed with 1% osmium tetroxide, and stained overnight with uranyl acetate. After dehydration with ethanol, the cell pellets were put in Spurr’s resin. The resin was cut into thin sections using an ultramicrotome (Leica EM UC7; Leica Microsystems GmbH, Wetzlar, Germany) and observed by TEM (Talos L120C; Talos).
The endosomal escape of nanoparticles was evaluated by assessing the cytoplasmic release of DNA from endolysosomes using confocal microscopy. To visualize the release of DNA, pSpCas9(BB) was labeled with Cy5 (Label IT Nucleic acid labeling kit; Mirus Bio LLC, Madison, WI, USA). The Cy5-labeled plasmid DNA was lipoplexed with MLNs or DPhPE-lacking MLNs, in which DPhPE was replaced with dipalmitoyl-phosphatidylcholine (Avanti Polar Lipids). At various time points after B16F10 cells were treated with lipoplexes, the cells were incubated with LysoTracker Green DND-26 (Thermo-Fisher Scientific) and DAPI (Sigma-Aldrich) for staining of endolysosomes and nuclei, respectively. The endosomal escape of the labeled plasmid DNA was monitored by confocal microscopy (LSM 710; Carl Zeiss, Jena, Germany).

Transfection Efficiency Measurement

Transfection efficiency was evaluated by measuring the fluorescence intensity from the expression of GFP-encoding plasmid DNA. First, 0.5 μg of pEGFP-n1 (Clontech) was complexed with MLNs at an N/P ratio of 7:1 to form the lipoplex, pGFP@MLN. For comparison, pGFP was complexed to Lipofectamine 2000 (Thermo-Fisher Scientific). B16F10 melanoma cells (Korean Cell Line Bank) were seeded in a 24-well plate at a density of 1 × 105 cells per well and incubated overnight. The B16F10 cells were then treated with various lipoplexes. After 12 h, the medium was replaced, and the transfected cells were incubated for 2 days. Cellular fluorescence was observed through a confocal microscope (TCS8; Leica Microsystems GmbH) and quantified by flow cytometry using a BD FACSCalibur (BD Biosciences, San Jose, CA, USA).

Measurement of Cell Viability and Exposure of Danger Signals on Tumor Cells

Cell viability and exposure of danger signals on tumor cells were tested after NIR irradiation of cells harboring the various nanoparticles. B16F10 melanoma cells were treated as indicated and irradiated for 5 min with 808 nm NIR light (1.5 W). The temperature was monitored using a FLIR T420 IR thermal imaging system (FLIR System Inc.). After NIR irradiation, the cells were seeded in a 24-well plate and incubated for 24 h. Cell viability was evaluated using the 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay. Live and dead cells were further visualized by calcein AM and PI staining (Molecular Probes, Eugene, OR, USA). Apoptosis of cells was measured by staining with annexin V and PI using an Annexin V apoptosis detection kit FITC (Thermo-Fisher Scientific). For measurement of DAMP molecules, calreticulin exposure on the cells was evaluated by flow cytometry. The NIR-irradiated cells were sequentially stained for 1 h with murine anti-calreticulin primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and Alexa Fluor 647-conjugated goat anti-mouse IgG secondary antibody (BioLegend, San Diego, CA, USA). The calreticulin-positive cell population was detected by flow cytometry using a BD FACSCalibur (BD Biosciences).

Calreticulin-Mediated Tumor Cell Phagocytosis by Dendritic Cells

Phagocytosis of tumor cells by dendritic cells was evaluated by coculture of dendritic cells with fluorescence-labeled B16F10 cells followed by flow cytometric analysis. Bone marrow-derived dendritic cells were isolated as previously reported. (56) After incubation for 7 days, dendritic cells were harvested and seeded on 24-well plates at a density of 1 × 105 cells per well. B16F10 cells treated with pGFP@MLNs were irradiated with NIR for 5 min and stained with CellTracker Red CMTPX Dye (Thermo-Fisher Scientific). The labeled B16F10 cells were cocultured with dendritic cells at a density of 1 × 104 cells per well for 8 h. The cells were then harvested, and dendritic cells were labeled with PE conjugated anti-CD11c antibody (BioLegend) and analyzed by flow cytometry.

Animals

All animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources at Seoul National University. The research protocol was approved by the Institutional Review Committee for the Use of Animals at the College of Pharmacy, Seoul National University (approval no. SNU-190821-5).

In Vivo Gene Editing Efficacy Test in a GFP-Expressing Cancer Model

In vivo gene editing was evaluated by molecular imaging of GFP-expressing cancer xenografts. GFP-expressing HeLa (HeLa-GFP; GenTarget Inc., San Diego, CA, USA) was chosen as a model cell line. HeLa-GFP cells were seeded in a 24-well plate at a density of 1 × 105 cells per well in complete DMEM. After overnight incubation, the cells were transfected with 1 μg/mL of plasmid DNA encoding Cas9 and GFP-targeting sgRNA (pC9sGfp) complexed with MLNs (pC9sGfp@MLNs) for 12 h. Plasmid DNA encoding Cas9 and scrambled sgRNA (pC9sScr) were used as a negative control. After 4 days of treatment, intracellular GFP fluorescence was observed through confocal microscopy and flow cytometry.
To test the efficiency of in vivo gene editing, 2 × 106 HeLa-GFP cells were mixed with 100 μL of matrigel (Corning Inc., Corning, NY, USA) and subcutaneously inoculated to the right flank of 6-week-old BALB/c nude mice (Raon Bio, Yongin, Republic of Korea). Seven days after inoculation, pC9sGfp@MLNs at a plasmid dose of 0.25 mg/kg were intratumorally injected twice at a 2-day interval. The fluorescence intensity at the tumor was measured with an IVIS Spectrum In Vivo Imaging System (PerkinElmer, Waltham, MA, USA). For fluorescence imaging of tumor tissue, the tumors were extracted and cryosectioned using a microtome (Leica CM3050 S cryostat; Leica Microsystems GmbH). The tissue sections were observed with an automated multimodal tissue analysis system (Vectra, PerkinElmer) and analyzed using the Inform software (PerkinElmer).

Measurement of TGF-β Gene Editing-Mediated Immune Response

Protein expression of TGF-β was evaluated by measuring TGF-β secreted to the culture medium of gene editing transfected B16F10 cells. B16F10 cells were seeded in a 24-well plate at a density of 1 × 105 cells per well. The next day, the cells were transfected with plasmid DNA coexpressing Cas9 and TGF-β-targeting sgRNA (pC9sTgf) complexed with MLNs (pC9sTgf@MLNs) for 12 h. Two days later, the medium was refreshed, and the cells were incubated for an additional 24 h. The concentration of TGF-β in the medium was then determined using a mouse TGF-β 1 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA).
The immune response induced by TGF-β gene editing was evaluated by monitoring the ability of T cells to undergo differentiation to FoxP3-expressing regulatory T cells (Treg cells) and assessing the immune suppressor function of decreasing expression of IFN-γ. The cell culture medium from TGF-β gene-edited B16F10 cells was incubated with splenocytes from 5-week-old C57BL/6 mice (Orient Bio, Inc., Seungnam, Republic of Korea). For differentiation of Treg cells, the medium was supplemented with 10 ng/mL of interleukin-2 (BD Biosciences) and 25 μL of Dynabeads Mouse T-Activator CD3/CD28 (Thermo-Fisher Scientific). After 48 h, the cells were stained with phycoerythrin (PE)-conjugated anti-CD4 (BioLegend), PE/Cy5-conjugated anti-CD25 (BioLegend), fluorescein isothiocyanate (FITC)-conjugated anti-CD127 (BioLegend), and allophycocyanin (APC)-conjugated anti-FoxP3 (Thermo-Fisher Scientific) antibodies for 1 h. The Treg population (CD4+ CD25+ CD127low FoxP3+) of the splenocytes was analyzed by flow cytometry. For the evaluation of Treg suppressor function, the splenocytes were stained with FITC-conjugated anti-CD3 (BioLegend), APC-conjugated anti-CD8 (BioLegend), and phycoerythrin-conjugated anti-IFN-γ (BioLegend) antibodies for 1 h. The IFN-γ expressing CD3+ CD8+ cell population in the splenocytes was analyzed by flow cytometry.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure the mRNA expression levels of perforin, granzyme B, CXCL10, FoxP3, and TGF-β in splenocytes. For RT-PCR, total RNA was extracted from splenocytes using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from mRNA using an AccuPower RT PreMix (Bioneer, Daejeon, Republic of Korea). qRT-PCR was performed using an Applied Biosystems Prism 7300 (Applied Biosystems, Waltham, MA, USA).

Evaluation of In Vivo Antitumor Efficacy in Primary Tumor, Secondary Tumor, and Metastasis Models

The in vivo antitumor efficacy of pC9sTgf@MLNs was evaluated in primary and rechallenged tumor models. Five-week-old C57BL/6 mice (Orient Bio, Inc.) were subcutaneously injected with 5 × 105 B16F10 cells in the right flank. Seven days after primary tumor inoculation, the mice were intratumorally injected with pC9sTgf@MLNs at a plasmid DNA dose of 0.25 mg/kg; this treatment was performed three times at 2-day intervals. One day after the last injection, the tumor site was irradiated with an 808 nm laser at a power of 1.5 W (BWT Beijing LTD) for 10 min, and the temperature at the tumor site was monitored using a FLIR T420 IR thermal imaging system (FLIR System Inc.). Tumor growth was monitored over 30 days from the primary tumor inoculation; the tumor volume was calculated as A × B2 × 0.5, where A and B are the lengths of the largest and smallest dimensions, respectively. (57)
To evaluate the antitumor immune responses against secondary tumors, 2 × 105 B16F10, MC-38 (ATCC, Manassas, VA, USA), or EL4 (Korean Cell Line Bank) cells were subcutaneously injected to the opposite flank 12 days after B16F10 primary tumor inoculation. The volume of the secondary tumor was monitored for 15 days from the tumor rechallenge.
For the lung metastasis model, 2 × 105 B16F10 cells were intravenously injected on day 12 after the primary tumor inoculation. At 33 days after the primary inoculation, mice were sacrificed and perfused with phosphate-buffered saline. Lung tissues were collected for tumor nodule counting and hematoxylin and eosin staining.

Evaluation of In Vivo TGF-β Genome Editing

In vivo gene editing of TGF-β in tumor tissue was analyzed by targeted deep sequencing. Five-week-old C57BL/6 mice (Orient Bio, Inc.) were subcutaneously inoculated with 5 × 105 B16F10 cells in the right flank. From 7 days after tumor inoculation, the mice were intratumorally injected three times with pC9sTg@MLNs (plasmid DNA dose of 0.25 mg/kg) in 2-day intervals. One day after the last injection, the tumor tissue was irradiated with an 808 nm laser at a power of 1.5 W (BWT Beijing LTD) for 10 min. After 8 h, the tumors were extracted and vigorously stirred with 2 mL of serum-free DMEM supplemented with 1 mg/mL of collagenase (Sigma-Aldrich) for single cell isolation. After 1 h, the tumor cell suspensions were centrifuged at 10 000 × g for 3 min. For indel analysis of dendritic cells (CD11c+), T cells (CD3+), and macrophages (CD11b+), the cell suspension from tumor tissue was stained with APC-conjugated anti-CD11c antibody (BioLegend), APC-conjugated anti-CD3 antibody (BioLegend), or APC-conjugated anti-CD11b (BioLegend) antibody. The genomic DNA was extracted from the collected cell pellets, and the TGF-β sgRNA target region (202 base pairs in length) was amplified by PCR using primers list in Table S1. Sequencing library construction was performed using TruSeq Nano DNA Kit (Illumina, San Diego, CA, USA), and samples were sequenced on Illumina HiSeq 2500 System (Illumina). The sequence data was analyzed using an Integrative Genomics Viewer (https://software.broadinstitute.org/software/igv/). (58)

In Vivo Immune Modulation Study in Tumor Microenvironment

The ability of TGF-β gene editing to modulate the tumor immune microenvironment was evaluated by assessing cytokine levels, tumor-infiltrating lymphocytes, and dendritic cell maturation. Five-week-old C57BL/6 mice (Orient Bio, Inc.) were subcutaneously injected with 5 × 105 of B16F10 cells in the right flank. After 7 days, the mice were intratumorally injected with pC9sTg@MLNs at a plasmid DNA dose of 0.25 mg/kg; this treatment was applied for a total of three times at 2-day intervals. One day after the last injection, the tumor tissue was irradiated with an 808 nm laser at a power of 1.5 W (BWT Beijing LTD) for 10 min. After 8 h, the tumors were extracted, weighed, and vigorously stirred with 2 mL of serum-free DMEM supplemented with 1 mg/mL collagenase (Sigma-Aldrich) for single cell isolation. After 1 h, the tumor cell suspensions were centrifuged at 10 000 × g for 3 min. The supernatant was collected for quantification of TGF-β and IFN-γ using ELISA kits (R&D Systems). The cytokine concentrations were normalized to 0.1 g of tumor tissue.
The remaining cell pellets were stained with FITC-conjugated anti-CD3 (BioLegend) and APC-conjugated anti-CD8 (BioLegend) antibodies for 1 h, and the cytotoxic T lymphocyte ratio in tumor tissues was assessed. For Treg cell staining, the cell pellets were resuspended and stained with PE-conjugated anti-CD4 (BioLegend), PE/Cy5-conjugated anti-CD25 (BioLegend), FITC-conjugated anti-CD127 (BioLegend), and APC-conjugated anti-FoxP3 (Thermo-Fisher Scientific) antibodies for 1 h and analyzed by flow cytometry. For dendritic cell staining, tumors and tumor draining lymph nodes were extracted. After single-cell preparation and centrifugation, the cell pellets were resuspended and stained with FITC-conjugated anti-CD11c antibody (BioLegend) and APC-conjugated anti-CD40 or APC-conjugated CD86 antibodies for 1 h and analyzed by flow cytometry. For effector memory CD8+ T cell staining, distant tumor draining inguinal lymph nodes were extracted and single-cell suspensions were prepared. The cells were stained for 1 h with FITC-conjugated anti-CD3 antibody, PerCP-Cy5.5-conjugated anti-CD8 antibody (BioLegend), PE-conjugated anti-CD44 antibody (BioLegend), and APC-conjugated anti-CD62L antibody (BioLegend) and then analyzed by flow cytometry.

In Vivo Safety Test

The in vivo safety of pC9sTgf@MLNs was evaluated by organ tissue staining, hematological and biochemical parameters, and body weights. Five-week-old C57BL/6 mice (Orient Bio, Inc.) were subcutaneously inoculated with 5 × 105 B16F10 cells in the right flank. From day 7 after tumor inoculation, the mice were intratumorally injected three times with pC9sTg@MLNs at a plasmid DNA dose of 0.25 mg/kg in 2-day intervals. One day after the last injection, the tumor site was irradiated with an 808 nm NIR laser at a power of 1.5 W (BWT Beijing LTD) for 10 min. Next day after the irradiation, mice were sacrificed, and major organs (heart, lung, liver, kidney and spleen) were extracted for hematoxylin and eosin staining. On the same day, as serum biochemical parameters, ALT, AST, and BUN values were measured using a clinical chemical analyzer (DRI-CHEM 2500s; Fujifilm, Tokyo, Japan), and hematological parameters of the whole blood were analyzed (NEODIN BioVet, Seoul, Republic of Korea). Body weights were measured over 20 days after tumor inoculation.

Biodistribution Kinetic Study

The time-dependent biodistribution profiles of pC9sTgf@MLNs were evaluated over 8 days postdose. Five-week-old C57BL/6 mice (Orient Bio, Inc.) were subcutaneously inoculated with 5 × 105 B16F10 cells in the right flank. On day 7 after tumor inoculation, the mice were intratumorally injected with pC9sTg@MLNs at a plasmid DNA dose of 0.25 mg/kg. At the indicated time points, tumor tissues and major organs were extracted and homogenized. The homogenates were further digested with aqua regia, and the Au content was quantified by ICP-MS (Varian 820-MS; Varian).

Cellular Uptake and Viability Test of MLNs in Normal and Immune Cells

In some experiments, lipoplex uptake and viability were evaluated in various normal cells, including bone marrow-derived dendritic cells, bone marrow-derived macrophages, splenic T cells, fibroblasts, and adipocytes. For isolation of bone marrow-derived macrophages, monocytes were collected from femurs and tibias of 5-week-old C57BL/6 mice and incubated for 7 days in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin, 100 units/mL penicillin, 20 ng/mL recombinant mouse M-CSF (GenScript), and 50 μM β-mercaptoethanol (Sigma-Aldrich). (59) Primary fibroblasts were isolated from the tails of 5-week-old C57BL/6 mice as reported previously. (60) Briefly, each excised tail tissue sample was digested with serum-free DMEM supplemented with 1 mg/mL collagenase (Sigma-Aldrich) and centrifuged at 1000 × g for 3 min. The pellet was maintained in complete DMEM. Adipocytes were differentiated from 3T3-L1 cells (Korean Cell Line Bank) as previously described. (61) Briefly, 3T3-L1 cells were maintained in complete DMEM supplemented with 1 mM dexamethasone (Sigma-Aldrich), 0.5 mM methylisobutylxanthine (Sigma-Aldrich), 1 μg/mL insulin (Sigma-Aldrich), and 2 mM rosiglitazone (Sigma-Aldrich) for 3 days. The medium was then changed to complete DMEM supplemented with 1 μg/mL insulin and maintained for 3 days.
For uptake and viability experiments, the various normal cells were seeded in a 24-well plate at a density of 1 × 105 cells per well. For the cellular uptake test, Cy5-labeled MLNs (fMLNs) were complexed with pC9sTgf to form pC9sTgf@fMLNs, which were applied to the cells for 6 h. The cells were then harvested, and the cellular uptake of pC9sTgf@fMLNs was analyzed by flow cytometry. For the viability test, the various normal cells were treated with pC9sTgf@MLNs for 6 h and irradiated for 5 min with 808 nm NIR light. Cell viability was evaluated using an MTT assay (Sigma-Aldrich).

In Vivo Nuclear Trafficking

DNA release and nuclear trafficking at tumor tissues were evaluated by detecting fluorescent dye-labeled DNA in nuclei isolated from tumor cells. Five-week-old C57BL/6 mice (Orient Bio, Inc.) were subcutaneously inoculated with 5 × 105 B16F10 cells in the right flank. On day 7 after tumor inoculation, the mice were intratumorally injected with the fluorescent Cy5-labeled pC9sTgf complexed to MLNs. At various time points, tumors were extracted, and nuclei were isolated using a Nuclei EZ Prep isolation kit (Sigma-Aldrich). The isolated nuclei were stained with DAPI, and the DNA fluorescence in the DAPI-positive population was measured by flow cytometry.
The mechanism by which DNA was released from MLNs was evaluated in a negatively charged model endosome system. (62) Endosome-like vesicles were prepared by thin-film hydration. Briefly, 0.5 μmol of l-α-phosphatidylglycerol (PG; Avanti Polar Lipids) and 2 μmol of l-α-phosphatidylcholine (PC; Avanti Polar Lipids) were dissolved in chloroform, and the organic solvent was removed under vacuum. For comparison, control vesicles were prepared with 2.5 μmol of l-α-phosphatidylcholine. The lipid thin film was hydrated with 0.5 mL of 20 mM HEPES buffer (pH 5.2). The resulting endosome-like vesicles were incubated with pC9sTgf@MLNs at a ratio of 5:1 (PG/EDOPC and DC-Chol) for 10 min at pH 5.2 and then analyzed by 1% agarose gel electrophoresis.

DNA Stability Test against Nuclease

DNA stability was evaluated by DNase I assay. pC9sTgf (0.5 μg) in naked form and complexed to MLNs was treated with 0.5 unit of DNase I (Sigma-Aldrich) for 30 min at 37 °C. The samples were then loaded on a 1% agarose gel, and DNA integrity was examined by electrophoresis.

Evaluation of Off-Target Effects

Off-target effects were assessed by measuring the levels of TGF-β gene editing and protein expression in major organs and lymph nodes. Five-week-old C57BL/6 mice (Orient Bio, Inc.) were subcutaneously inoculated with 5 × 105 B16F10 cells in the right flank. Beginning on day 7 after tumor inoculation, the mice were intratumorally injected three times with pC9sTg@MLNs at a plasmid DNA dose of 0.25 mg/kg at 2-day intervals. One day after the last injection, the tumor site was irradiated with an 808 nm NIR laser at a power of 1.5 W (BWT Beijing LTD) for 10 min. On days 13 and 30 after tumor inoculation, major organs (heart, lung, liver, kidney, spleen) and inguinal lymph nodes were extracted, weighed, and vigorously stirred with 2 mL of serum-free DMEM supplemented with 1 mg/mL of collagenase (Sigma-Aldrich) for single-cell isolation. After 1 h, the single-cell suspensions were centrifuged at 10 000 × g for 3 min. Genomic DNA was extracted from the cell pellets and tested for the occurrence of TGF-β gene editing using T7E1 endonuclease. The supernatant was collected for quantification of TGF-β using an ELISA kit (R&D Systems). The obtained concentrations were normalized to 0.1 g of the source tissue. For assessment of cytokine release syndrome, blood samples were collected at days 13 and 30 after tumor inoculation, and the serum levels of IL-6, C–C motif chemokine ligand 2 (CCL2), and C-reactive protein (CRP) were measured with ELISA kits (R&D Systems).

Evaluation of Autoimmune Responses

For assessment of autoimmune responses, the serum levels of anti-nuclear antibody and anti-dsDNA antibody were measured. Five-week-old C57BL/6 mice (Orient Bio, Inc.) were subcutaneously inoculated with 5 × 105 B16F10 cells in the right flank. Beginning on day 7 after tumor inoculation, the mice were intratumorally injected three times with pC9sTg@MLNs at a plasmid DNA dose of 0.25 mg/kg, at 2-day intervals. One day after the last injection, the tumor site was irradiated with an 808 nm NIR laser at a power of 1.5 W (BWT Beijing LTD) for 10 min. On days 13 and 30 after tumor inoculation, blood was collected. The serum levels of anti-nuclear antibody were quantified by ELISA (MyBioSource, San Diego, CA, USA). For anti-dsDNA antibody detection, 96-well EIA/RIA assay microplates (Corning Inc.) were coated with 0.1 mg/mL of poly l-lysine (Sigma-Aldrich) for 30 min and then with DNA from calf thymus (Sigma-Aldrich) for 30 min. The wells were blocked with 2% BSA, and the plates were incubated with 1 mg/mL horseradish peroxidase-conjugated rat anti-mouse IgG for 2 h. The 3,3′,5,5′-tetramethylbenzidine substrate was added, and absorbance was measured at 450 nm with a SpectraMax M5 plate reader (Molecular Devices).

Statistics

Statistical analysis of experimental data was conducted using two-sided analysis of variance (ANOVA) with the Student–Newman–Keuls post hoc test, which was applied by the SigmaStat software (version 12.0; Systat Software, Richmond, CA, USA). A p-value less than 0.05 was considered statistically significant.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications Web site at DOI: xxx. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.1c05420.

  • sgRNA and primer sequences, schematic illustration of nanoparticle preparation, characterization and zeta potential of nanoparticles, physical stability, effect of MLN composition on intracellular trafficking of plasmid, temperature change and cell viability without NIR irradiation, cellular apoptosis by lipoplex treatment and NIR irradiation, phagocyotosis of cancer cells by dendritic cells, gene editing efficacy in GFP model, off-target effect of TGF-β gene editing, flow cytometry gating strategy, mRNA expression levels of inflammatory or immunosuppressive markers, effect of dosing frequency and NIR irradiation regimens on NIR responsiveness, in vivo photothermal efficacy without NIR irradiation, NIR irradiation effect on untreated tumors, antitumor efficacy of MLN-mediated TGF-β editing without NIR irradiation, the appearance of the MLN-treated mice without NIR irradiation, in vivo indel frequency of pC9sTgf@MLN(+)-treated tumor, population of effector memory T cells in distant tumor draining lymph nodes, population of immune cells in tumor microenvironment, appearance of extracted lung after metastatic tumor challenge, histology of major organs, biochemical and hematological parameters, body weights, stability of pC9sTgf@MLNs against nuclease, nuclear trafficking of plasmid in tumor tissues, release of plasmid from MLNs, biodistribution profile of pC9sTgf@MLNs, and pC9sTgf@MLN uptake and viability of various cells (PDF)

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Author Information

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  • Corresponding Authors
    • Gayong Shim - School of Systems Biomedical Science, Soongsil University, Seoul 06978, Republic of Korea Email: [email protected]
    • Yu-Kyoung Oh - College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of KoreaOrcidhttps://orcid.org/0000-0002-0969-3339 Email: [email protected]
  • Authors
    • Dongyoon Kim - College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
    • Yina Wu - College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This research was funded by grants from the National Research Foundation, Ministry of Science and ICT, Republic of Korea (NRF-2021R1A2B5B03002123; NRF-2018R1A5A2024425; NRF-2020R1I1A1A01070084), Ministry of Education, Republic of Korea (NRF-2021R1A6A3A01086428), and the Korean Health Technology R&D Project (No. HI15C2842, HI18C2177, HI19C0664), Ministry of Health & Welfare, Republic of Korea.

References

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  • Abstract

    Figure 1

    Figure 1. Construction of pC9sTgf@MLNs and proposed mechanisms for its restructuring of the tumor immune microenvironment. (A) Cationic MLNs containing Au metal clusters were prepared by reducing Au3+ ion with ascorbic acid. For Cas9/sgRNA-mediated gene editing of TGF-β, pC9sTgf was complexed to MLNs to form pC9sTgf@MLN lipoplexes. (B) TGF-β contributes to the immune suppressive tumor microenvironment through diverse signaling on various immune cells. pC9sTgf@MLN-mediated TGF-β gene editing restructures the tumor immune microenvironment to an “immune-activated” state. The phototherapy-induced exposure of the “eat-me” signal will enable tumor antigen uptake by activated dendritic cell and induce the tumor antigen-specific immune memory system.

    Figure 2

    Figure 2. Physicochemical features of MLNs. (A) Schematic illustration of MLNs with reduced elemental Au inside. (B) TEM image of MLNs. Scale bar = 50 nm. (C) Elemental composition of MLNs was analyzed by high-angle annular dark-field (HAADF) microscopy and energy-dispersive X-ray spectroscopy-scanning TEM (STEM-EDS). Elemental Au and P are shown in green and red colors, respectively. Scale bar = 50 nm. (D) Contents of elemental Au and P in nanoparticles measured by phosphate assay and inductively coupled plasma-mass spectrometry (ICP-MS), respectively (n = 5 per group). (E) Light absorbance spectra of nanoparticles measured from 300 to 1000 nm. (F) Colors and thermal images after NIR irradiation for LNs and MLNs. (G) Temperature of samples monitored during three times of repeated cycles of 808 nm NIR laser irradiation at a power of 1.5 W/cm2 with 4 min pulse and 4 min pause. (H) Gel retardation of pDNA@MLNs complexed at various N/P weight ratios observed on a 1% agarose gel. (I) Size distribution of MLNs or pDNA@MLNs measured by dynamic light scattering.

    Figure 3

    Figure 3. Cellular uptake and intracellular fate of plasmid DNA and nanoparticles. (A) B16F10 cells were treated with lipoplexes of red dye labeled DNA with MLNs for various durations and stained with LysoTracker (green color). The intracellular fate of DNA was monitored by confocal microscopy for 24 h. Scale bar = 10 μm. (B) Fluorescence signal of GFP was observed under confocal microscopy. Scale bar = 250 μm. (C) Transfected cells were analyzed by flow cytometry (n = 3 per group). (D) pGFP@MLN-treated cells were visualized by dark-field microscopy. Scale bar = 250 μm. (E) Au content of the cells was measured by ICP-MS (n = 3 per group). (F) Endocytosis of pGFP@MLNs was observed in B16F10 cells by TEM imaging (n.s., not significant; ***p < 0.001).

    Figure 4

    Figure 4. NIR responsiveness and exposure of calreticulin. The temperatures of B16F10 cells transfected with various nanoparticles were monitored by thermal imaging during NIR irradiation. The highest temperature reached by each group was visualized (A) and plotted (n = 3 per group) (B). One day after this treatment, the viability of B16F10 cells was visualized by live (green) and dead (red) cell staining (C) and by MTT assay (D) (n = 5 per group). (E) Cellular apoptosis was detected by annexin V and PI staining (n = 5 per group). (F) The exposure of calreticulin was analyzed by flow cytometry. The calreticulin-positive cell populations (G) and mean fluorescence intensity (MFI) (H) were quantified (n = 3 per group; ***p < 0.001).

    Figure 5

    Figure 5. Gene-editing ability of pC9sGfp@MLNs. (A) To visualize the gene-editing ability, pC9sGfp was delivered by MLNs to GFP-stable cells. (B) Fluorescence signal of the HeLa-GFP cells was observed by confocal fluorescence microscopy after various transfections. (C) GFP-edited cells were analyzed by flow cytometry. (D) Efficiency of GFP gene editing was examined by T7 endonuclease 1 (T7E1) assay. A blue arrow indicates the amplified product of the target region, and red arrows indicate the cleavage products. (E) HeLa-GFP-xenografted mice received pC9sGfp@MLNs twice at a 2-day interval. (F, G) One day after the last transfection, the fluorescence signals of tumors were observed by molecular imaging (n = 3 per group). (H) Tumors were sectioned for fluorescence microscopy (*p < 0.05; **p < 0.01).

    Figure 6

    Figure 6. MLN-mediated gene editing effect on secretory TGF-β. (A) TGF-β-edited B16F10 cells are expected to exhibit reduced secretion of TGF-β. (B) Sequence structure of pC9sTgf and the sgRNA binding position in exon 1 of the TGF-β gene. (C) Expression of Cas9 protein in nanoparticle-treated cells was confirmed by Western blot analysis. (D) Representative sequencing analysis of polymerase chain reaction (PCR) amplicons of target region. A red arrow indicates a cleavage site and dotted lines indicate the deleted region. (E) TGF-β editing efficiency was observed by T7E1 endonuclease assay. (F) Concentration of secretory TGF-β in pC9sTgf@MLN-treated B16F10 cells was measured by ELISA (n = 5 per group; ***p < 0.001).

    Figure 7

    Figure 7. T cell differentiation by MLN-mediated TGF-β editing. (A) B16F10 cells were treated with various groups of nanoparticles, and the supernatants were applied to splenocytes. Two days later, the populations of Treg cells and IFN-γ+ CD8+ cells were analyzed. (B) Populations of FoxP3+ Treg cells were tested by flow cytometry. (C) Populations of IFN-γ+ CD8+ cells were tested by flow cytometry. Critical gates were marked with boxes. (D, E) Quantified cell populations were plotted for Treg cells (D) and IFN-γ+ CD8+ cells (E) (n = 5 per group; *p < 0.05; ***p < 0.001).

    Figure 8

    Figure 8. Antitumor efficacy of MLN-mediated TGF-β editing. (A) B16F10-bearing C57BL6 mice were intratumorally injected with various lipoplexes in the three tumor immune microenvironments with a 2-day interval. One day after the third injection, tumor sites were irradiated with NIR. (B) Thermal images of NIR-irradiated mice were obtained using a thermal camera. (C) Temperatures of tumor sites with or without NIR irradiation were plotted (n = 5 per group; ns, not significant; ***p < 0.001). (D) Tumor volumes of various groups were monitored for over 27 days after tumor inoculation. (E) Appearance of mice treated in various groups was observed at 18 days after tumor inoculation.

    Figure 9

    Figure 9. Activation of the tumor immune microenvironment by MLN-mediated TGF-β editing. (A) B16F10-bearing C57BL6 mice received three injections of pC9sTgf@MLNs at 2-day intervals. One day after the last transfection, tumor tissues were collected for analysis. (B) In vivo gene editing efficiency was measured by T7E1 endonuclease assay. (C) Representative indel sequences of target regions were analyzed by targeted deep sequencing. Red arrow indicates the cleavage site, and dotted lines indicate the deleted region. (D) Concentration of TGF-β was measured in the secretome obtained from tumor cells (n = 5 per group). (E) Populations of Treg cells, cytotoxic T cells, and mature dendritic cells in the tumor tissues were analyzed by flow cytometry. Critical gates were marked with boxes. Quantified cell populations were plotted for Treg cells (F), cytotoxic T cells (G), and mature dendritic cell (H) (n = 5 per group). (I) Concentration of IFN-γ was measured in the secretome from the tumor cells (n = 5 per group; ***p < 0.001).

    Figure 10

    Figure 10. Transcriptomic analysis and immune cell populations in the tumor microenvironment. (A) B16F10-bearing C57BL6 mice were treated with the various lipoplexes three times at 2-day intervals. One day after the last injection, tumor sites were irradiated by NIR. Tumor tissues were collected and RNA from CD3+ cells was used for next-generation sequencing transcriptomic analysis. (B) Tumor tissues were stained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclei (blue) and fluorescent dye-tagged anti-TGF-β antibodies (red). Scale bar = 2 mm. (C) Tumor tissues were stained with DAPI for nuclei (blue), fluorescent dye-tagged anti-TGF-β antibodies (red), fluorescent dye-tagged anti-CD8 antibodies (yellow), fluorescent dye-tagged anti-FoxP3 antibodies (sky blue), and fluorescent dye-tagged anit-CD31 antibodies (green). Scale bar = 50 μm.

    Figure 11

    Figure 11. Antitumor effect of MLN-mediated TGF-β editing. (A) B16F10 tumor-bearing C57BL6 mice received pC9sTgf@MLNs or other lipoplexes three times at a 2-day interval. One day after the last transfection, tumors were irradiated with NIR, and mice were rechallenged with B16F10 tumor cells in the opposite flank. Volume of the rechallenged tumor (B) and survival of mice (C) were monitored. (D, E) Tumor draining lymph nodes were collected for analysis on day 13. Single-cell suspensions from the lymph nodes were stained with the dendritic cell maturation markers, CD40 (D) and CD86 (E), and analyzed by flow cytometry. Critical gates were marked with boxes. (F, G) Populations of CD11c+CD40+ cells (F) and CD11c+CD86+ cells (G) were quantified and plotted (n = 5 per group). (H) Populations of effector memory CD8+ T cells (CD3+CD8+CD44+CD62Llow) in distant tumor lymph nodes were quantified and plotted (n = 5 per group; ***p < 0.001).

    Figure 12

    Figure 12. Antitumor efficacy against B16F10, MC38, and EL4 distant tumors and B16F10 lung metastasis. (A) B16F10-bearing C57BL6 mice were injected with pC9sTgf@MLNs three times at 2-day intervals. One day after the last injection, tumors were irradiated with NIR, and mice were rechallenged with B16F10, MC38, or EL4 tumor cells in the opposite flank. (B) Tumor volumes were monitored over 30 days after the primary B16F10 inoculation. (C) Appearance of mice was observed on day 27 after the primary tumor inoculation. (D) B16F10-bearing C57BL6 mice were treated with pC9sTgf@MLNs three times at 2-day intervals. One day after the last injection, tumors were irradiated with NIR and mice were intravenously rechallenged with B16F10. (E) Three weeks after the secondary B16F10 injection, lung tissues were sectioned and subjected to hematoxylin and eosin staining. (F) Numbers of B16F10 nodules in lungs were quantified (n = 5 per group; ns, not significant; ***p < 0.001).

    Figure 13

    Figure 13. Lack of off-target effects or autoimmune responses. B16F10 tumor-bearing mice were intratumorally injected with pC9sTgf@MLNs three times at 2-day intervals. In the pC9sTgf@MLN(+) group, the mice were irradiated with NIR 1 day after the last injection. (A) On days 13 and 30 after tumor inoculation, major organs and lymph nodes were extracted from mice. TGF-β gene editing was evaluated by T7E1 assay. (B) TGF-β levels were quantified by ELISA (n = 5 per group). (C) Serum levels of IL-6, CCL2, and CRP were analyzed by ELISA (n = 5 per group). (D, E) On days 13 and 30, blood samples were extracted from naive mice and pC9sTgf@MLN(+)-treated mice, and anti-nuclear antibody (ANA) (D) and anti-dsDNA antibody (E) were analyzed by ELISA (n = 5 per group; ns, not significant).

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  • Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications Web site at DOI: xxx. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.1c05420.

    • sgRNA and primer sequences, schematic illustration of nanoparticle preparation, characterization and zeta potential of nanoparticles, physical stability, effect of MLN composition on intracellular trafficking of plasmid, temperature change and cell viability without NIR irradiation, cellular apoptosis by lipoplex treatment and NIR irradiation, phagocyotosis of cancer cells by dendritic cells, gene editing efficacy in GFP model, off-target effect of TGF-β gene editing, flow cytometry gating strategy, mRNA expression levels of inflammatory or immunosuppressive markers, effect of dosing frequency and NIR irradiation regimens on NIR responsiveness, in vivo photothermal efficacy without NIR irradiation, NIR irradiation effect on untreated tumors, antitumor efficacy of MLN-mediated TGF-β editing without NIR irradiation, the appearance of the MLN-treated mice without NIR irradiation, in vivo indel frequency of pC9sTgf@MLN(+)-treated tumor, population of effector memory T cells in distant tumor draining lymph nodes, population of immune cells in tumor microenvironment, appearance of extracted lung after metastatic tumor challenge, histology of major organs, biochemical and hematological parameters, body weights, stability of pC9sTgf@MLNs against nuclease, nuclear trafficking of plasmid in tumor tissues, release of plasmid from MLNs, biodistribution profile of pC9sTgf@MLNs, and pC9sTgf@MLN uptake and viability of various cells (PDF)


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