Novel Polymers in microparticulate diagnotic agents

Polychelating amphiphilic polymers to optimize the concentrations of contrast agents used in medical diagnostic imaging.

Vladimir P. Torchilin

Medical diagnostic imaging requires that an appropriate intensity of a certain signal from an area of interest be achieved to differentiate this area from surrounding tissues, whatever imaging technique is used. Imaging involves the relationship between the three spatial dimensions of the region of interest and a fourth dimension, time, which relates to the pharmacokinetics of the imaging agent and the period necessary to acquire the image (1). Currently used medicalimaging techniques include

-imaging (the application of -emitting radioactive materials);
magnetic resonance (MR, a phenomenon based on the transition between different energy levels of atomic nuclei under the influence of a radio frequency signal);
computed tomography (CT, the use of ionizing radiation with the aid of computers to acquire cross-images of the body and 3-D images of areas of interest); and
ultrasonography (US, the use of irradiation with ultrasound based on the different rates at which ultrasound passes through various tissues).

See also the Diagnostic substances for imaging methods for the chemical species, or "reporters", used with each of these techniques. Nonenhanced imaging techniques are useful only when relatively large tissue areas are involved in the pathological process. Although attenuations - the abilities of tissues to absorb various signals (e.g., X-rays, sound waves, ionizing radiation, and radio frequencies) - differ, in most cases these differences are not sufficient for clear discrimination between the normal and pathological regions. To achieve sufficient attenuations, contrast agents are used that can absorb certain types of irradiation much more strongly than the surrounding tissues. The objective is to accumulate a sufficient quantity of a contrast agent in the area of interest and to minimize its presence in normal tissues and organs.

The differing chemical characteristics of the reporter groups and their different signal intensities result in wide variations in the tissue concentration that must be achieved for successful imaging (Table 1). The concentration is rather low for -imaging and noticeably higher for MRI and CT. For this reason, the natural progression in the development of effective contrast agents led to the use of microparticulate carriers for the efficient delivery of multiple agents to areas of interest, thus enhancing signals from these areas.

Liposomes and micelles
Liposomes (microscopic artificial phospholipid vesicles) and micelles (amphiphilic compound colloidal particles with lipophilic cores and hydrophilic coronas) are preferred as contrast agent carriers because of their easily controlled properties and good pharmacological characteristics (excellent biocompatibility and biodegradability). For specific in vivo delivery, the sizes, charges, and surface properties of these carriers can be easily changed by adding new ingredients to the lipids or amphiphilic compound mixtures before the liposomes or micelles are prepared and by varying the preparation methods (2). Figure 1 illustrates the formation and structures of micelles and liposomes and the ways that various reporter species are covalently or noncovalently incorporated into different compartments of these carriers.

The ability of liposomes to entrap different substances into the aqueous phase and the membrane compartment makes them suitable for carrying the diagnostic reporters used with all four imaging techniques (3, 4). The varying chemical properties of reporter compounds require specific protocols for loading the liposomes. In addition, the imaging techniques differ not only in their sensitivity and resolution, but also in the amounts of diagnostic labels to be delivered into the areas of interest. With these considerations in mind, we developed various liposome preparation methods, along with the relatively new approach of micellar transport of contrast agents (5). In this discussion, I concentrate on contrast liposomes and micelles for - and MR imaging (i.e., on liposomes and micelles loaded with radioactive and nonradioactive heavy metals).

When phospholipid liposomes are introduced into the circulatory system, they are rapidly sequestered by the cells of the reticuloendothelial system (RES) (half-clearance time <30 min). Liver cells are primarily responsible for absorbing the liposomes (6), and the sequestration is almost independent of the size, charge, and composition of the liposomes. RES uptake can be somewhat decreased by using small liposomes, increasing liposome dose (blocking the RES), presaturating the RES with "empty" liposomes or other particles, or modifying the liposome surface with certain "protective" polymers. To increase the quantity of liposomes accumulating in the "required" areas, I have suggested the use of targeted liposomes (liposomes with a surface-attached, specific ligand) (7). Liposomes with a specific affinity for an affected organ or tissue are believed to increase the efficacy of liposomal pharmaceutical agents, including those used for imaging. Immunoglobulins, primarily of the class G (IgG), are the most promising and widely used targeting species for various drugs and drug carriers, including immunoliposomes.

Still, the pharmacokinetic properties of immunoliposomes (and other targeted immunoparticulates) in many cases are not satisfactory. Despite the apparent success in the development of antibody-to-liposome coupling techniques and improvements in targeting efficacy, the majority of immunoliposomes are absorbed by the liver, usually because of insufficient time for interaction between the target and targeted liposome. This is especially true when the target of choice has a diminished blood supply (ischemic or necrotic areas), and only a few blood-borne liposomes pass through the target while they are still in the circulatory system. The same insufficient targeting occurs if the concentration of the target antigen is very low, and the number of productive collisions between target antigens and immunoliposomes is too small. Clearly, much better accumulation can be achieved if liposomes can remain in circulation longer.

So far, the best method to prolong circulation is to coat liposome surfaces with inert, biocompatible polymers, such as poly(ethylene glycol) (PEG), which form a protective layer over the surface and retard liposome recognition by opsonins (substances that bind to antigens, triggering an attack by phagocytes) and subsequent removal from the blood (8). Long-lived liposomes are now widely used in biomedical in vitro and in vivo studies and have even been introduced into clinical practice (9). The use of PEG-coated liposomes and micelles as carriers of contrast agents has also become an important research area (4, 5).

An important analysis of the pharmacokinetics of PEG-coated liposomes was performed by Allen et al. (10). They determined that the most important results of drug incorporation into liposomes are delayed drug absorption, restricted drug biodistribution, delayed drug removal, and retarded drug metabolism. All of these objectives are achieved by entrapping the drug in a liposome or other carrier, which hinders interstitial penetration of the drug and decreases accessibility to the biosystem in general. The presence of a protective polymer on the carrier surface further improves its performance. Thus, even though "plain" liposomes (and micelles) have nonlinear, saturable kinetics, long-lived liposomes demonstrate dose-independent, nonsaturable, and log-linear kinetics. These properties are typical for all long-circulating drugs and carriers, including imaging agents.

Loading carriers with contrast agents
As seen in Figure 1, contrast agents can be loaded only into the core of a micelle, whereas they can reside in the internal water compartment and the membrane of a liposome. The relative efficacy of entrapment of radio-opaque materials into different liposomes as well as the advantages and disadvantages of various liposome types were analyzed by Tilcock et al. (11). -Imaging and MR imaging require liposomes containing sufficient quantities of radionuclides and paramagnetic metals, respectively. Two methods are frequently and efficiently used to prepare liposomes for - and MR imaging. In the first, the metal is converted to a soluble chelate (using an agent such as diethylenetriaminepentaacetic acid [DTPA]) and then introduced into the aqueous interior of a liposome (12). In the second, DTPA or a similar chelating agent may be derivatized with a lipophilic group, which can anchor it to the liposome surface during or after liposome preparation (13). Different chelating agents and lipophilic anchors were tested for the preparation of ¹¹¹In-, ^99mTc-, Mn-, and Gd-treated liposomes (13, 14).

Because most clinically relevant radioisotopes have rather short half-lives (<3 days), the last step of the preparation of contrast liposomes for -imaging takes place immediately before application. Therefore, liposomes must be prepared that can be sufficiently loaded with a contrast agent with a simple and fast labeling protocol. Phillips and Goins have written a good review of the problems associated with the use of liposomes for -imaging (15).

In MR imaging, the quality of the resonance signal is maximized if the reporter atoms are exposed for interaction with the aqueous phase. This requirement makes encapsulation of the metal into the liposome less attractive than if the metal coupled with chelating agent was exposed to water. In addition, low-molecular-weight, water-soluble paramagnetic probes may leak from liposomes upon contact with body fluids, which destabilizes most liposomal membranes. Moreover, Tilcock has shown that when excessive concentrations of Gd-DTPA are encapsulated inside liposomes to improve signal enhancement, the relaxivity (intensity of MR signal) of the compound might be lower than for the nonencapsulated Gd-DTPA complex, probably because of a decreased residence lifetime of water molecules inside the vesicles (16). Membranotropic chelating agents such as DTPA-stearylamine and DTPA-phosphatidylethanolamine consist of a polar head that contains a chelated paramagnetic atom and a lipid moiety that anchors the metal-chelate complex to the liposome membrane. In terms of the relaxivity of the final formulation, this technique is far superior to liposome-encapsulated paramagnetic ions because of the decrease in the rotational correlation time of the paramagnetic species rigidly connected to the relatively large particle (16). Liposomes with membrane-bound paramagnetic ions also have a smaller risk of leakage into the body. Membranotropic chelates are suitable for micelle incorporation (they anchor to the lipophilic micelle core), and they also may be suitable for loading micelles with heavy radio metals (Figure 1).

Chelating polymers
Chelating polymers have been used to increase the load of diagnostic antibodies with reporter radioactive or paramagnetic metals (17). To couple multiple reporter groups to a single antibody molecule without inactivating it by overmodification with too many individual chelate residues (e.g., DTPA), Slinkin et al. suggested the use of a polymer chain carrying many chelating groups as side chains (18). Such chelating agents provide high-affinity binding of a wide range of heavy metal ions. The polymeric backbone for attachment of multiple chelating agents has to contain a sufficient number of reactive groups, such as amino, carboxy, aldehyde, or sulfhydryl. Natural and synthetic polymers can be used; poly-l-lysine (PLL), containing multiple free amino groups (one per unit), is the most frequent choice (19). To couple the chelating agent to a PLL, the reactive intermediate of the chelate, containing a mixed anhydride, cyclic anhydride, N-hydroxysuccinimide ester, tetrafluorophenyl ester, isothiocyanate, or the like, is usually used.

For the subsequent interaction with a protein (antibody) to be labeled, the chelating polymer is activated by any available method, such as carbodiimide treatment or thiolation, and then reacted with the protein to form an amide or thioether bond. Bifunctional reagents, such as N-succinimidyl 3-(2`-pyridyldithio)propionate (SPDP) or succinimidyl maleidomethylcyclohexanecarboxylate (SMCC), can also be used successfully. To avoid the formation of cross-linked products and minimize the modification of the biological properties of a protein, my group has suggested and experimentally designed a new scheme for chelating-polymer-antibody conjugation involving the preparation of a chelating polymer with a single terminal reactive group capable of interaction with an antibody (18, 19). The approach is based on the use of carbobenzoxy (Cbz)-protected PLL with a free terminal amino group, which is derivatized into a reactive form with subsequent deprotection and incorporation of DTPA. This process can involve the use of activating bifunctional agents such as SPDP and SMCC. Figure 2 shows the chemistry of a typical preparation of a single-terminus-activated chelating polymer (via SPDP in this particular case). Multiple pathways for conjugating such polymers with an antibody antigen-binding fragment (Fab) or any other protein (via protocols that use SPDP, SMCC, or bromoacetylation) are given in Figure 3.

As the result of such modifications, it is possible to achieve very high loadings of reporter metals in antibodies. Up to several dozen atoms of radioactive (¹¹¹In) or MR-active (Gd) metals can be firmly attached to a single protein molecule, compared with just a few metal atoms that can be attached to traditional monomeric chelates (19, 20).

Liposomes and micelles with PAPs
Because it is rather difficult to enhance the signal intensity from a reporter metal in liposomes and micelles, we attempted to increase the quantity of metals in carriers by using polychelating polymers modified with lipophilic groups to ensure their firm incorporation into the liposome membrane or micelle core. Thus, we developed PAPs (polychelating amphiphilic polymers), a new family of copolymers. PAPs contain a hydrophilic fragment with multiple chelating groups and a relatively short but highly lipophilic phospholipid fragment on one end of the polymer chain that is suitable for incorporation into liposomes and micelles. The approach is again based on the use of Cbz-protected PLL with a free terminal amino group, which is derivatized into a reactive form with subsequent deprotection and incorporation of DTPA (21). However, in this case, using the PLL N-terminus modification chemistry, we developed a pathway for the synthesis of the amphiphilic polychelate N,-(DTPA-polylysyl)glutarylphosphatidylethanolamine (DTPA-PLL-NGPE).

This polychelate easily assimilates into the liposomal membrane or micelle core in the preparation process and sharply increases the number of chelated heavy metal atoms attached to a single lipid anchor (Figure 1). This increases the number of bound reporter metal atoms per vesicle and decreases the dosage of an administered lipid without compromising the image signal intensity. The PAP synthesis - a single-terminus-activated chelating polymer preparation and the attachment of a lipophilic anchor to it - is described in Figure 4. Upon incorporation into the bilayer, the NGPE anchor grafted with a chelating polymer forms a "coat" of chelating groups around the liposomal membrane or within the outer shell of a micelle. In the case of MR, metal atoms chelated into these groups are directly exposed to the aqueous environment that enhances the relaxivity of the paramagnetic ions and, consequently, the vesicle contrast properties. The enhanced relaxivity of liposomes containing Gd-loaded PAP compared with membranotropic single chelating groups, at the same mole fraction of chelate-associated lipophilic anchors, is clearly shown in Figure 5. In addition, liposomes can be modified with PEG to improve stability and longevity in the biosystem.

Micelles formed by self-assembled amphiphilic polymers (such as PEG-phosphatidylethanolamine, PEG-PE) can also be loaded with amphiphilic PLL-based chelates carrying diagnostically important metal ions such as ¹¹¹In and Gd (22). The final preparations are quite stable in vivo. Upon subcutaneous administration, the micelles penetrate the lymphatic system and effectively allow its elements to be visualized with the appropriate imaging methods. The micelles remain primarily in the lymph fluid rather than accumulating in the nodal macrophages (because of the protective effect of the surface PEG groups) and move rapidly through the lymphatic system. Micelles are fast and efficient lymphangiographic agents for imaging and MR imaging.

To illustrate the performance of contrast PAP-modified liposomes and micelles, I have shown images of normal and pathological components of the lymphatic system obtained in rabbits with Gd-polychelate liposomes (Figure 6). Gd-loaded amphiphilic polychelate (Gd-DTPA-PLL-NGPE)-containing liposomes with an average diameter of 215 nm were subcutaneously injected into the forepaw of a sedated rabbit, and images were acquired. The high content of Gd in the liposomes due to the use of a membrane-incorporated amphiphilic chelating polymer permitted fast and informative diagnostic visualization of a VX₂ sarcoma in a rabbit popliteal lymph node; the tumor was clearly seen 10 min after injection. In comparison, the time between contrast agent administration and image acquisition for other lymphotropic imaging agents is usually several hours. Similar experiments with PEG-PE mixed micelles with core-incorporated, Gd-loaded amphiphilic chelates provided fast and efficient visualization of different sections of the lymphatic system (Figure 7).

We used Gd-polychelate-loaded liposomes coated with PEG to do experimental MR angiography in dogs. This long-lived MR imaging agent is under commercial development by Biostream Inc. Gd-DTPA-PLL-NGPE/PEG-liposome preparation containing >30 wt % Gd was intravenously injected into a normal 11-kg dog, and MR imaging was performed. Because the blood half-life for the preparation was experimentally found to be ~2 h, the imaging session was conducted for 1 h. Within this period, all major vessels remained clearly delineated (Figure 8). The preparation also made it possible to visualize much smaller vessels such as the renal artery, carotid arteries, and jugular veins.

The future of PAPs
Perhaps the key benefit of liposome- and micelle-based diagnostics is the ability to image and measure functional characteristics of a region of interest in the body. If we understand the characteristics of the microparticulate carrier and how it interacts with the biological system, it should be possible to design diagnostics that make these functional measurements possible. Contrast-agent-loaded liposomes and micelles will be successfully used for visualization of numerous organs, tissues, and disease sites with all imaging methods. The advantages of - and MR imaging with liposomes (and more recently with micelles) have been already demonstrated in many experimental and clinical studies. -Imaging with contrast liposomes and micelles seems to be the most attractive method because of the relatively small quantities of carriers and reporter metals needed for successful imaging, sharply minimizing side effects. By using polymeric chelates, liposomes and micelles can be easily loaded with sufficient quantities of radioactive or MR-active agents. In addition, grafted PEG can serve as a capture-avoiding agent, permitting effective accumulation of the diagnostic label in the target.

Before liposomes and micelles become the imaging agents of choice, several important questions must be answered. We need to accomplish these objectives in the near future:

learn much more about the pharmacokinetics of liposomal and micellar imaging agents,

find a way to increase the carrier loading with reporters to minimize overloading the patient with the carrier and to decrease the cost of preparation,

learn more about the possible toxicity of metal-loaded liposomes and micelles caused by intracellular release of metals, and

achieve cost-effective large-scale production of homogeneous preparations of diagnostic liposomes and micelles.

References

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(15) Phillips, W. T.; Goins, B. In Handbook of Targeted Delivery of Imaging Agents, Torchilin, V. P., Ed.; CRC Press: Boca Raton, FL, 1995; pp 149-173.

(16) Tilcock, C. In Liposome Technology, 2nd ed.; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1993; Vol. 2, pp 65-87.

(17) Torchilin, V. P.; Klibanov, A. L.; Nossiff, N. D.; Slinkin, M. A.; Strauss, H. W.; Haber, E.; Khaw, B. A. Hybridoma 1987, 6, 229-240.

(18) Slinkin, M. A.; Klibanov, A. L.; Torchilin, V. P. Bioconj. Chem. 1991, 2, 342-348.

(19) Torchilin, V. P.; Klibanov, A. L. CRC Crit. Rev. Ther. Drug Carriers Syst. 1991, 7, 275-308.

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(22) Trubetskoy, V. S.; Frank-Kamenetsky, M. D.; Whiteman, K. R.; Wolf, G. L.; Torchilin, V. P. Acad. Radiol. 1996, 3, 232-238.

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