MULTIFUNCTIONAL SYNTHETIC POLY(L-GLUTAMIC ACID)-BASED CANCER THERAPEUTIC AND IMAGING AGENTS

Abstract

Modern polymer chemistry has led to the generation of a number of biocompatible synthetic polymers have been increasingly studied as efficient carriers for drugs and imaging agents. Synthetic biocompatible polymers have been used to improve the efficacy of both small-molecular-weight therapeutics and imaging agents. Furthermore, multiple targeted anticancer agents and/or imaging reporters can be attached to a single polymer chain, allowing multifunctional and/or multimodality therapy and molecular imaging. Having both an anticancer drug and an imaging reporter in a single polymer chain allows noninvasive real-time visualization of the pharmacokinetics of polymeric drug delivery systems, which can uncover and explain the complicated mechanisms of in vivo drug delivery and their correlation to pharmacodynamics. This review examines use of the synthetic biocompatible polymer poly(L-glutamic acid) (PG) as an efficient carrier of cancer therapeutics and imaging agents. This review will summarize and update our recent research on use of PG as a platform for drug delivery and molecular imaging, including recent clinical findings with respect to PG-paclitaxel (PG-TXL); the combination of PG-TXL with radiotherapy; mechanisms of action of PG-TXL; and noninvasive visualization of in vivo delivery of polymeric conjugates with contrast-enhanced magnetic resonance imaging (MRI), optical imaging, and multimodality imaging.

1. INTRODUCTION

Over the past two decades, synthetic polymers have been increasingly studied as efficient carriers for drugs and imaging agents. Modern polymer chemistry has led to the generation of a number of biocompatible polymer structures, including branched (1), graft (2, 3), and multivalent polymers (4) and dendrimers (5, 6). These biomedical macromolecules, which vary in size ranging from 10–1000 nm (7), have unique pharmacokinetic properties, including prolonged blood circulation, enhanced tissue retention, and preferential accumulation in lesions with leaky vasculature (8). For these reasons, polymers have since been used as effective drug delivery devices (7, 9). Drugs or any therapeutic agent of interest are normally encapsulated, adsorbed, or conjugated on the surface (10). Delivery of these drugs are accomplished by utilizing the natural characteristics of the tissue or organ, such as the leaky vasculature of tumors, drugs encapsulated within the macromolecule, are then delivered through enhanced permeability and retention effect (passive targeting) (8). Active targeting of a therapeutic agent, in contrast with passive targeting, is achieved by conjugating the therapeutic agent or the carrier system to a tissue or cell-specific ligand (10, 11).

Synthetic biocompatible polymers have been used to improve the efficacy of both small-molecular-weight therapeutics and imaging agents (12). Conjugation of small molecular weight anticancer agents to biocompatible polymers increases deposition of drug at the tumor site and reduces harmful effects of the drug on normal tissues. Conjugation of imaging agents to biocompatible polymers prolongs the reporters’ blood circulation time and therefore increases their concentration in the blood, with increased concentrations, which allows imaging of the vasculature with a higher vessel-to-background signal ratio. Furthermore, multiple targeted anticancer agents and/or imaging reporters can be attached to a single polymer chain, allowing multifunctional and/or multimodality therapy and molecular imaging (13). Having both an anticancer drug and an imaging reporter in a single polymer chain allows noninvasive real-time visualization of the pharmacokinetics of polymeric drug delivery systems, which can uncover and explain the complicated mechanisms of in vivo drug delivery and their correlation to pharmacodynamics. Combining drug delivery and molecular imaging in one macromolecular platform also allows simultaneous detection and treatment of disease. This results in more efficient and effective therapeutic regimens, more accurate detection and diagnosis, rapid and noninvasive assessment of response to therapy, and personalized patient care.

This review examines use of the synthetic biocompatible polymer poly(L-glutamic acid) (PG) as an efficient carrier of cancer therapeutics and imaging agents. The chemistry and applications of PG and of PG conjugates with various chemotherapeutic agents were previously reviewed (14, 15). In this review, we will summarize and update our recent research on use of PG as a platform for drug delivery and molecular imaging, including recent clinical findings with respect to PG-paclitaxel (PG-TXL); the combination of PG-TXL with radiotherapy; mechanisms of action of PG-TXL; and noninvasive visualization of in vivo delivery of polymeric conjugates with contrast-enhanced magnetic resonance imaging (MRI), optical imaging, and multimodality imaging.

2. POLYMER-DRUG CONJUGATES

Historically, stumbling blocks in cancer drug development have included dose-limiting toxic effects, limited aqueous solubility, in vivo instability, and nonselectivity. In the past, much effort was devoted to developing novel formulations that would ensure the injectability, stability, and safety of anticancer drug candidates. Today, among the novel formulations being investigated are polymer-drug conjugates.

In the mid-1970s, Ringsdorf proposed a model for a polymer–drug conjugate that could enhance the delivery of an anticancer drug to a tumor (16). In this model, a polymeric carrier is conjugated with a drug to enhance its pharmacologic properties, and a homing ligand can also be attached for active targeting (Figure 1A). Since then, polymer–drug conjugates have become a fast-growing field, and nearly a dozen polymer–drug conjugates have advanced to the clinical trial stage. Results from early clinical trials of the polymer–drug conjugates have demonstrated several advantages over the corresponding parent drugs, including fewer side effects, enhanced therapeutic efficacy, ease of drug administration, and improved patient compliance. Enhanced therapeutic efficacy is achieved primarily through the enhanced permeability and retention (EPR) effect of long-circulating polymers (8). To date, several synthetic polymers have been successfully advanced into clinical trials studies or have been introduced into clinical practice, including polyethylene glycol (PEG) (17–20), poly- styrene–maleic anhydride copolymer (SMA) (21, 22), N-(2-hydroxypropyl)–methacrylamide copolymer (HPMA) (23–27), cyclodextrin (28, 29), and poly(L-glutamic acid) (PG) (30–33). Excellent reviews have documented the use of these polymers in cancer therapy (19, 34–38) and further review of these conjugates is beyond the scope of the current review and will only focus on PG-drug conjugates.

Figure 1.

(A) Model for targetable polymer–drug conjugate according to Ringsdorf (16). A solubility enhancer, a pharmacokinetic modifier, a homing device, and specific drugs or imaging probes can be attached to the same polymeric chain (adapted from (66)). (B) of PG-TXL.

Unlike other synthetic polymers that have been tested in clinical studies, which have a nondegradable C–C backbone, PG is composed of naturally occurring L-glutamic acid linked together through amide bonds (Figure 1B). The pendant free γ carboxyl group in each repeating unit of L-glutamic acid is negatively charged at a neutral pH, which renders the polymer water soluble. The carboxyl groups also provide functionality for drug attachment. PG is also biodegradable (39). These features make PG a promising candidate for polymer–drug conjugates for selective delivery of chemotherapeutic agents. PG–drug conjugates accumulate and are retained in solid tumors through the EPR effect (8). Uptake of PG–drug conjugates in target cells may also be increased by specific receptor-mediated interactions of PG–drug conjugates containing targeting ligands.

Doxorubicin (40–42), daunorubicin (43), 1-β-D-araninofuranosylcytosine (Ara-C) (44), uracil and uradine (45), cyclophosphamide (46), L-phenylalanine mustard (47), and mitomycin C (48) have all been conjugated to PG and practically all of them have shown better antitumor activity than the parent drug. However, these conjugates have not advanced to clinical trials and detailed studies of these conjugates are needed in order to fully assess the potential in treating solid tumors. Camptothecin and paclitaxel conjugated to PG are the only PG-drug conjugated that has advanced to clinical trials. Camptothecin was conjugated directly into PG through ester bonds (49) or with glycine linker (50). It has been found out that when camptothecin is directly conjugated to PG, the maximum achievable drug payload achievable is 15% due to steric hindrance but with the glycine linker, the loading could reach as high as 50% by weight (51). Increasing the molecular weight of PG (from 33kDa to 50 kDa) and increasing the loading of camptothecin also increased antitumor efficacy without substantially altering the maximum tolerated dose (51).

3. PG–PACLITAXEL

PG–paclitaxel (PG-TXL) (Opaxio^™, formally known as CT-2103, Xyotax^®) has advanced to phase III clinical trials and is positioned to be the first synthetic polymer–drug conjugate to reach the market.

3.1. Preclinical Antitumor Activity

Paclitaxel is a mitotic spindle inhibitor that stabilizes microtubules and, as a result, interferes with essential cellular functions such as mitosis, cell transport, and cell motility (52). Paclitaxel has shown remarkable antineoplastic effect against a wide range of human cancers, including ovarian cancer, non-small cell lung cancer, head and neck cancer, melanoma, and Kaposi’s sarcoma (53). It was commercially developed by Bristol-Myers Squibb and was first sold under the trademark Taxol^™. In this formulation, Taxol^™ is administered with a mixture of polyethoxylated castor oil (Cremophor) and ethanol as a vehicle, and is infused over 3 to 24 h. Taxol^™ is now used to treat patients with lung, ovarian, breast, head and neck cancer, and advance forms of Kaposi’s sarcoma (54–57). Taxol^™ is also used for the prevention of restinosis (58). A newer formulation, under the trademark Abraxane^™, is paclitaxel bound to human albumin. This formulation is infused in 30 mins and does need to treat the patients with contraindications for allergy as they would in Taxol^™. Abraxane has been approved in January 2005 for treatment of breast cancer after failure of combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy (59–62).

PG-TXL was initially developed to overcome the poor aqueous solubility of paclitaxel and avoid hypersensitivity reactions associated with the use of paclitaxel formulated with Cremophor and ethanol. In PG-TXL, paclitaxel is conjugated to PG through its 2′-hydroxyl group via an ester linkage (Figure 1B). The resulting conjugate is highly water soluble (>20 mg/kg) and has demonstrated significantly enhanced antitumor efficacy and improved safety compared with free paclitaxel in preclinical studies (63). For example, in a syngeneic murine OCa-1 ovarian carcinoma model, a single dose of PG-TXL in saline (10 mg equivalent paclitaxel/ml) at a dose of 80 mg equivalent paclitaxel/kg caused significant tumor growth delay compared with tumor growth with the same dose of paclitaxel in Cremophor and ethanol (63). Furthermore, a complete tumor cure was observed in all 26 mice injected with PG-TXL at its maximum tolerated dose (MTD) of 160 mg equivalent paclitaxel/kg, which is twice the MTD of free paclitaxel. Similarly, in Fischer rats with well-established rat 13762F breast adenocarcinoma, treatment with PG-TXL caused complete tumor eradication at PG-TXL’s MTD (60 mg equivalent paclitaxel/kg) and also at a lower dose (40 mg equivalent paclitaxel/kg) (12). In contrast, free paclitaxel at its MTD (40 mg/kg) caused only a transient growth delay of approximately 15 days (Figure 2A).

Figure 2.

(A) Antitumor activity of free paclitaxel and PG-TXL in rats bearing 13762F breast tumors. Each drug was injected intravenously in a single dose at the indicated equivalent paclitaxel concentration. Data are presented as means ± standard deviations of tumor volume (adapted from Ref. (63)). (B) Effect of PG-TXL on radiocurability of OCA-I tumor after single or fractionated irradiation. Mice bearing 7-mm tumors in the right hind leg were given i.v. 80 mg/kg PG-TXL and/or local tumor irradiation with graded doses of β-rays delivered as a single dose or as daily fractions for 5 consecutive days. When the two agents were combined, PG-TXL was given 24 h before the start of irradiation. Radiation-dose–response curves were generated for local tumor control at 120 days after treatment with single-dose irradiation alone (△), fractionated irradiation (○), PG-TXL plus single-dose irradiation (▲), or PG-TXL plus fractionated irradiation (●). Error bars are 95% confidence intervals on the TCD50 (adapted from Ref. (71)).

The antitumor activity of PG-TXL was further evaluated in mice with different syngeneic murine tumors (MCa-4 and MCa-35 breast cancer, HCa-1 hepatocellular carcinoma, and FSa-II sarcoma; intramuscular inoculation), mice with human SKOV3ip1 ovarian tumor cells injected intraperitoneally, and nude mice with orthotopic human MDA-MB-435Lung2 breast tumors in mammary fat pads (64, 65). All PG-TXL treatments were given intravenously as a single bolus injection. All the murine tumors, whether sensitive or resistant to paclitaxel treatment, showed significant growth delay with PG-TXL at its MTD (160 mg equivalent paclitaxel/kg) and at lower doses. In mice with human SKOV3ip1 tumors, PG-TXL significantly extended survival. Median survival was 75 days for mice treated with PG-TXL at 120 mg equivalent paclitaxel/kg, 56 days for mice treated with paclitaxel, and 59 days for mice treated with Cremophor plus ethanol vehicle. In mice with human MDA-MB-435Lung 2 tumors, PG-TXL at 120 mg equivalent paclitaxel/kg produced tumor regression in 50% of the cases. In the remaining mice, with progressively growing tumors, micrometastases in the lung were found in only 25% of the animals. In contrast, paclitaxel at 60 mg/kg did not produce tumor regression, and metastases were found in 42% of the mice (64). In another study (65), nude mice bearing intraperitoneally implanted NMP-1 and HEY ovarian tumors, which were paclitaxel resistant, showed significant improvement in survival and even some cures after a single intraperitoneal treatment with PG-TXL (65). These studies demonstrated that PG-TXL has significant therapeutic activity against a broad range of solid tumors and metastases.

3.2 Clinical Findings

Results of clinical trials PG-TXL were summarized in the literature by Li and Wallace (66). The formulation of PG-TXL used in clinical studies has a median molecular weight of PG-TXL equal to 48,000 Da, and about 37% paclitaxel by weight, which is equivalent to approximately one paclitaxel molecule for every 11 glutamic acid units in each PG polymer chain (67). This formulation obviates the use of Cremophor and ethanol and allows infusion of paclitaxel as PG-TXL over 30 min every 3 weeks with maximum tolerated dose of 177 mg/m² (68).

The clinical trials reported to date show that compared with conventional treatment with free paclitaxel, treatment with PG-TXL as a single agent produces similar or better survival and is less toxic. In these trials, PG-TXL showed three safety-related advantages. First, alopecia was rare, and complete hair loss was not observed. Second, nausea and vomiting were uncommon. Third, hypersensitivity reactions were rare, and those that did occur were usually mild to moderate; thus, routine use of prophylactic premedications was not required (66). PG-TXL has been shown to be active against a variety of cancers (66).

The studies with PG-TXL to date have mostly been conducted in patients who have already received multiple courses of chemotherapy with other agents. Further studies to define toxicity and efficacy in patients with less prior therapy are needed to determine the role of PG-TXL in these cancers as first-line and second-line therapy. Of particular note is the observed improvement in overall survival for women less than 55 years old or women with normal estrogen levels receiving PG-TXL in combination with carboplatin versus women receiving standard chemotherapy, in combined log-rank analysis of the STELLAR 3 and STELLAR 4 phase III trials. Consequently, a confirmatory phase III trial was initiated for the treatment of chemotherapy-naïve advanced non-small cell lung cancer (NSCLC) in women with estradiol greater than 25 pg/comparing PG-TXL plus carboplatin with paclitaxel plus carboplatin. The primary objective of this study is to compare the overall survival of patients randomized to the PG-TXL/carboplatin arm to that of patients randomized to the comparator arm, paclitaxel/carboplatin. Secondary objectives are to compare the progression-free survival, disease control, clinical benefit, response rate, quality of life, and the safety and tolerability of the treatment arms (http://www.celltherapeutics.com).

3.3 Use for Radiosensitization

It has previously been shown that in patients with many types of solid tumors, the combination of chemotherapy and radiotherapy results in significantly improved response and survival rates compared with the rates observed with chemotherapy and radiotherapy alone.

Chemotherapy agents can act as radiosensitizers, enhancing the tumor response to radiation. The antitumor effect of combined PG-TXL and radiotherapy was evaluated in C3Hf/Kam mice with syngeneic OCa-1 ovarian tumors and MCa-4 mammary carcinoma model use either tumor growth delay or fifty percent tumor cure as the end points. PG-TXL has demonstrated tumor radiation enhancement factors from 4.0 to 8.0 as compared to 1.5 to 2.0 for paclitaxel (69–71) (Figure 2B). PG-TXL has a stronger radiosensitizing effect than paclitaxel owing to sustained release of the free drug from polymer-bound drug in the tumor, which is partly attributed to the EPR effect of the macromolecules. Irradiation can in turn potentiate the tumor response to polymer–drug conjugates by increasing tumor vascular permeability and thus the uptake of these conjugates into solid tumors (70).

These promising preclinical studies prompted the initiation of a phase I clinical trial of PG-TXL combined with concurrent radiotherapy and a phase I PG-TXL in combination with cisplatin and radiotherapy in patients with esophageal by the Brown University Group (72, 73). On the basis favorable phase I data, a single-arm phase II study was initiated in 40 patients with esophageal cancer using neoadjuvant PG-TXL, cisplatin, and radiation regiment. Three of 40 patients (7.5%) achieved a complete clinical endoscopic response and refused surgery. Twelve of the 37 patients who underwent surgery (32%) achieved a complete pathologic complete response. There was no treatment related death. Only one patient required a feeding tube and one patient required total parenteral nutrition. In comparison, the pathologic complete response rate with chemotherapy (cisplatin/5-FU)/radiation in patients with esophageal adenocarcinoma is approximately 25% (74, 75). The rate of grade 3/grade 4 esophagitis with 5-FU/cisplatin/radiation in esophageal cancer is 42% (76). Thus, PG-TXL/cisplatin plus radiation is a well tolerated, easily administered regimen for esophageal cancer. The treatment resulted in a low incidence of significant esophagitis and demonstrated a high pathologic complete response rate.

3.4 Mechanism of Action

In vitro, both paclitaxel and PG-TXL induce extensive telomere erosion and telomeric associations, which are the early manifestations of apoptosis (77). Morphological analysis and biochemical characterizations in a panel of breast cancer cell lines confirmed that paclitaxel and PG-TXL had similar abilities to induce apoptosis, independent of p53 status. Flow cytometric analysis further revealed that both paclitaxel and PG-TXL induced a characteristic G2/M arrest in the cell cycle (78). However, compared with paclitaxel, PG-TXL appears to have reduced potency in vitro (63). These data are consistent with disturbance of microtubule polymerization being the major mechanism of action for PG-TXL and suggest that the release of paclitaxel or active species from PG-TXL is required for PG-TXL to exert its action.

In vivo, PG-TXL showed a biodistribution pattern different from that of free paclitaxel (69). On the basis of area under the tissue concentration–time curve values, tumor exposure to paclitaxel was five times greater with PG-TXL than with paclitaxel formulated in Cremophor-EL-plus-ethanol vehicle. PG-TXL was retained much longer than free paclitaxel in tumors because of slower elimination of the conjugate. Furthermore, in another study in mice, the concentration of free paclitaxel released from PG-TXL remained relatively constant in tumor tissue over a period of 144 h, whereas the concentration of free paclitaxel in tumor tissue of mice injected with paclitaxel in Cremophor-EL-plus-ethanol vehicle was reduced more than sixfold by 144 h after injection (79). Singer et al. (67) showed that compared with paclitaxel, PG-TXL had a prolonged blood half-life and negligible release of the active agent, paclitaxel (~1% of the total taxane). Thus, the EPR effect of PG-TXL is most likely an important contributing factor responsible for the superior therapeutic index of PG-TXL observed in preclinical studies.

In addition to the EPR effect, PG-TXL also mediates selective degradation and release of paclitaxl in tumor tissues. Using high-performance liquid chromatography/mass spectral analysis, Shaffer et al. (80) compared the profiles of PG-TXL metabolites in murine RAW 264.7 monocyte/macrophage-like cells and in cancer cells (NCI-H460 lung cancer cells and HT-29 colon cancer cells). They showed that the major intracellular metabolites of PG-TXL resulting from the degradation of polymer backbone were monoglutamyl-2′-paclitaxel (Glu-2′-TXL) and diglutamyl-2′-paclitaxel (H2N-Glu-Glu-2′-TXL). Significantly, the intracellular concentrations of Glu-2′-TXL and H2N-Glu- Glu-2′-TXL were 100 to 1000 times higher in the RAW 264.7 cells than in the cancer cells (80).

Recent studies suggest that selective proteolysis of the PG backbone through the action of cellular proteases in tumors, particularly cathepsin B, may also be responsible for the increased site-specific delivery and enhanced antitumor activity of PG-TXL (80, 81). Cathepsin B is a lysosomal cysteine protease found in normal cells and tissues. In premalignant and malignant lesions, the expression of cathepsin B is highly upregulated, and the enzyme is secreted and becomes associated with the cell surface (82). Cathepsin B has been implicated in degradation of the extracellular matrix, a crucial step in tumor dissemination and angiogenesis. Using a noninvasive near-infrared (NIR) fluorescence optical imaging technique, we demonstrated selective degradation of L-PG polymer by cysteine proteases, such as cathepsin B and cathepsin L (Figure 3A), selective degradation of L-PG versus D-PG (Figure 3B), and selective inhibition of L-PG degradation in the presence of cathepsin B inhibitor (Figure 3C) (81). This fluorescence optical imaging technique can be very useful in investigating the spatial and temporal distributions of the degradation products of PG-based polymeric drugs and the effect of polymer degradation on the antitumor activity of polymer–drug conjugates.

Figure 3.

Enzymatic degradation of L-PG-NIR813. (NIR813 is a dye that fluoresces and absorbs at 813 nm.) (A) Kinetics of polymer degradation by cathepsin B. Various concentration of L-PG-NIR813, from 0.625 to 25 μM, were incubated with 0.2 units of cathepsin B at 25°C. (B) Selective degradation of L-PG-NIR813 (8%) versus D-PG-NIR813 in the presence of 0.8 unit/mL cathepsin B at incubation time of 4h at 37°C. (C) Inhibition of L-PG-NIR813 degradation by cathepsin B inhibitor II. L-PG-NIR813 (8% loading; 10 μM equivalent NIR813) was incubated with increasing concentrations of cathepsin B inhibitor II and 0.2 units of cathepsin B and imaged at 24 h. No L-PG-NIR813 was added in control wells (adapted from (81)).

Finally, recruitment of macrophages residing in tumors is a possible mechanism responsible for the intratumoral dispersion of PG-TXL. Distribution to necrotic zone/hypoxia may be responsible to radiosensitization effect of PG-TXL (see Section 4.3 for details).

4. PG CONJUGATES AS IMAGING AGENTS

Over the past two decades, synthetic polymers have been increasingly studied as carriers for contrast agents. Compared with nonpolymeric imaging agents, polymeric imaging agents have prolonged plasma half-lives, enhanced stability, reduced toxicity, and improved targeting and nonspecific binding, thereby allowing for more specific and amplified imaging that can significantly differentiate target areas from background. Additionally, multiple reporters and/or homing ligands can be attached to a synthetic polymer, allowing multimodality cancer imaging.

As previously mentioned, PG is a polymeric carrier widely used for diagnosis and therapeutics because of its biocompatibility, biodegradability, and water-solubility (66). PG also has prolonged blood circulation time, and with PG, multiple functions can be combined in a single polymeric chain. When drugs and imaging agents are combined in a single PG polymer, imaging with different modalities can elucidate the mechanism and possible mode of action of PG-drug conjugates.

4.1 PG for MRI

PG conjugated with diethylenetriaminepentaacetic acid gadolinium chelate (PG-DTPA-Gd) was developed in our laboratory as a biodegradable blood pool MRI contrast agent. It is synthesized through a simple two-step reaction. First, p-aminobenzyl-DTPA-t-butyl ester is attached to the side chain of PG via an amide bond. Second, the protective t-butyl ester is removed in the presence of trifluoroacetic acid, and gadolinium is chelated in sodium acetate buffer, pH 5.2. PG-DTPA-Gd is a narrow-distribution polymer conjugate with molecular weight of ~100K Da and polydispersity of 1.23.

PG has been shown to be degraded by cysteine proteases (83). In vitro, conjugates of PG, such as PG-DTPA-Gd, were demonstrated to be degraded within 24 h in the presence of cathepsin B (83). In vivo, PG-DTPA-Gd, as an MRI blood pool imaging agent, showed enhanced vascular contrast in mice up to 2 h after contrast injection. By 24 h after injection, the blood-pool activity had largely returned to the precontrast level (Figure 4A–B). Interestingly, the contrast in certain regions of the tumors was clearly enhanced at 24 h after contrast injection, reflecting the EPR effect (84) of macromolecules in solid tumors. In vivo biodistribution studies in mice confirmed the gradual clearance of the contrast agent from the blood pool and kidney (85). Similar findings were confirmed in rhesus monkeys.

Figure 4.

(A–B)Whole-body MR images of mice before and at 10 min, 2 h, and 24 h after intravenous injection of PG-DTPA-Gd at a dose of 0.04 mmol Gd/kg. Lu: lung; Li: liver; IVC, inferior vena cava; K, kidney; T: tumor (adapted from (85)).Note: A and B denotes different coronal slice to show the different organs.(C) Representative magnetic resonance images of OCA-1 tumors from an untreated mouse (a–e) and a mouse treated with PG-TXL (120 mg equivalent paclitaxel/kg) (f–j). Transverse T1-weighted spin echo images before (a, f) and at 10 min (b, g), 2 days (c, h), and 4 days (d, i) after intravenous injection of PG-DTPA-Gd (0.04 mmol/kg). (e, j) Macroscopic photographs of the tumors sectioned on the transverse plane showing the necrotic regions in the tumors (N = necrosis; T = tumor; M = muscle). Arrows in b and g: peripheral tumor enhancement owing to blood pool imaging effect of the contrast agents; arrowheads in d: necrotic area. (k) Higher-power photomicrographs (left panel: hematoxylin and eosin stain; right panel: factor VIII immunohistochemical stain) of tumor in (j) from boxed region. Note the scattered factor VIII–positive cells in the necrotic zone (arrows). Scale bar: 100 μm (adapted from (85)).

Intratumoral distribution of PG-DTPA-Gd was further investigated in mice bearing human Colo-205 xenografts or syngeneic murine OCA-1 ovarian tumors. MRI of tumors revealed specific accumulation of the polymeric contrast agent in necrotic tumor tissue (Figure 4C). Although the mechanism of the selective localization of PG-DTPA-Gd in areas of necrosis is not entirely clear, the observation that biotin-PG-DTPA-Gd co-localized with macrophages in OCA-1 tumors suggests that the affinity of PG-DTPA-Gd for necrotic tissue may be mediated through macrophages (86). The contrast enhancement in necrotic tumor tissue was observed only with polymeric contrast agents, not with low-molecular-weight agents. High-molecular-weight contrast agents have prolonged blood circulation times, which may give them sufficient time to interact with migrating or resident macrophages. The EPR effect of macromolecules also played a role in their transport from the vicinity of tumor blood vessels to the necrotic areas through a diffusion process.

These findings suggest that PG is an essential carrier for gadolinium contrast moieties to tumor necrotic tissue. Consistent with this hypothesis, studies using a monoclonal antibody for paclitaxel that specifically recognizes PG-TXL showed that PG-TXL was taken up by macrophages in vitro through endocytosis.

Another PG-based MR imaging agent is PG conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid gadolinium chelate (PG-DO3A-Gd), which was synthesized by Ye and co-workers (87). DO3A is the derivative of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) in which one methylcarboxylic arm is modified by 1,6-hexanediamine, a linker with which Gd-DO3A is attached to PG. The in vivo properties of PG-DO3A-Gd was tested using polymer conjugates with molecular weights of 87, 50, and 28 kDa (87). MR images of mice bearing MDA-MB-231 human breast cancer xenografts were acquired continuously for the first 20 min and then at 30, 60, 120, 180, 240, 360, and 1440 min postinjection. The conjugate with the lowest molecular weight (28 kDa) rapidly cleared from the circulation and had lower tumor accumulation, whereas the conjugates with higher molecular weights (50, 87 kDa) exhibited more prolonged blood circulation and higher tumor uptake (87). Following this study, Vaidya and collegues conjugated PG-DO3A-Gd with a photosensitizer, mesochlorin e₆ (Mce₆) for monitoring and mediating photodynamic therapy (PDT) (88). Results from this study showed that PG-DO3A-Gd-Mce₆, a bifunctional polymer conjugate containing both an MRI contrast agent and a photosensitizer, was effective for cancer imaging (contrast-enhanced MRI) and efficacious for treatment with PDT in an animal model. PG-DO3A-Gd-Mce₆ was recently modified with polyethylene glycol (PEG) to improve its efficacy in contrast-enhanced (CE)-MRI-guided PDT by reducing non-specific liver uptake and increasing tumor accumulation (89). The pegylated conjugate demonstrated longer blood circulation, less liver uptake, and more tumor accumulation than the non-pegylated conjugate, as shown by MRI. Site-directed laser irradiation of tumor resulted in higher therapeutic efficacy for the pegylated conjugate. Moreover, animals treated with PDT and pegylated PG-DO3A-Gd-Mce₆ showed reduced vascular permeability on dynamic CE-MRI and decreased microvessel density in histological analysis (89). Lu et al. (90) reported their study of a PG-DO3A-Gd conjugate linked with a degradable cystamine spacer. The conjugate was designed to solve the problem of slow excretion of macromolecular contrast agents and potential toxicity due to release of the Gd(III) ion caused by metabolism of the agents. Studies in nude mice bearing OVCAR-3 human ovarian carcinoma xenografts demonstrated that the conjugate produced significant MRI blood pool contrast enhancement and pharmacokinetic MRI study showed that the Gd(III) chelate from the conjugate accumulated in the urinary bladder in a kinetic pattern similar to that of Omniscan^™ or Gd-diethylenetriaminepentaacetic-acid-bis-methylamide (Gd(DTPA-BMA)), suggesting that the chelate was released by the endogenous thiols and excreted through renal filtration (90).

The peptide, arginine-glycine-aspartic acid (RGD), was conjugated to PG-DO3A-Gd-cystamine to target α_νβ₃ integrin, an angiogenesis biomarker in neoplastic tissues (91). In vitro and in vitro binding studies using DU145 cells, which highly express α_νβ₃ integrin, revealed that the binding affinity of polymer-bound cyclic-RGD-phenylalanine-lysine, or c(RGDfK), had higher binding compared with SLK cells, which was consistent with free c(RGDfK) (91). In vivo MR imaging showed a significant decrease in the T₁ values of water protons in the periphery of DU145 tumors, while no significant decrease in T₁ values was observed in SLK tumors, as shown on the MR T₁ maps (91). These results demonstrate that the conjugation of RGD peptide to PG-DO3A-Gd-cystamine resulted in a specific affinity for the human prostate carcinoma DU145 cell line and was effective for the detection of the angiogenesis biomarker α_νβ₃ integrin in the DU145 tumor with quantitative T₁ mapping. The targeted polymeric Gd(III) chelate conjugate with a degradable spacer has the potential to be a new paradigm for safe and effective probes in molecular imaging.

4.2 PG for Optical Imaging

Optical imaging is an emerging method for the detection and monitoring of cancer. Optical imaging has high sensitivity, permits rapid data acquisition, and involves relatively inexpensive instrumentation. Imaging of radiation in the NIR region of the electromagnetic spectrum (wavelengths between 650 and 900 nm) is highly desirable because most biological substances and impurities in bioprocesses absorb and fluoresce between 190 and 650 nm. In contrast, in the ultraviolet-visible region, the relative sensitivity in the detection of biomolecules is greatly reduced owing to the high background noise caused by the molecules themselves (autofluorescence) and the impurities within the ultraviolet-visible region (92). Since the NIR region is a region of the spectrum beyond these effects, it provides high sensitivity in the detection of biomolecules and an improved signal-to-noise ratio.

Optical imaging has provided insights into how PG-TXL is degraded in tumors. As discussed previously, PG-TXL has been shown to have significantly higher uptake in tumors and greater antitumor activity than the parent drug, paclitaxel (63, 64). One of the proposed mechanisms for the increased site-specific delivery and enhanced antitumor activity of PG-TXL is degradation of the PG backbone through the action of cellular proteases in tumors, which releases paclitaxel into the tumors. To understand PG’s in vivo degradation, an inexpensive NIR813 fluorescent dye containing a primary amine was conjugated to the pendant –COOH groups of PG having a molecular weight of ~60 kDa (81). Interestingly, the fluorescence intensity decreased with increasing amounts of NIR813 dye (Figure 5A). NIR loading of 8% was optimal to start with an optically silent PG-NIR, but upon exposure to cathepsin B (an enzyme that is overexpressed in most tumors), the fluorescence intensity increased 10-fold. This phenomenon was tested in mice bearing U87 human glioma tumors. Figure 5B shows that L-PG-NIR813, but not D-PG-NIR813, was degraded in tumors with overexpression of cathepsin B. These results indicate that PG-NIR may be used to monitor the in vivo degradation of L-PG-based polymeric drugs and that this agent may be useful in noninvasive imaging of protease activity, particularly that of cysteine proteases (81).

Figure 5.

(A) Effect of NIR813 loading on degradation of L-PG-NIR813 in the presence of cathepsin B. Aliquots (100 mL) of each L-PG-NIR813 solution (10 mM equivalent NIR813) in 20 mM sodium acetate buffer (pH 5.2) were incubated with cathepsin B (0.4 units/ml) at 37°C for 24 h in triplicate (adapted from (81)). (B) Representative in vivo near infrared fluorescence (NIRF) images of cathepsin B activity in intracranially inoculated U87/TGL tumors. The mice used in the study had the same tumor burden, as indicated by the bioluminescence signal generated after intravenous injection of luciferin. NIRF images were acquired 24 h after intravenous injection of L-PG-NIR813 (50 nmol/mouse) with the same NIR dye loading (10%) and the same molecular weight (17,500). The NIRF signal was visualized in the mouse injected with L-PG-NIR813 but not the mouse injected with D-PG-NIR813 (adapted from (81)).

4.3 PG for Multimodality Imaging

We have conducted several investigations of PG polymers that permit both optical imaging and MRI. While optical imaging offers high sensitivity in the detection of tumors, MRI offers excellent spatial resolution and intrinsic soft-tissue contrast.

The combination of optical imaging and MRI has the potential to identify lymph node metastases noninvasively and significantly improve the safety of sentinel lymph node (SLN) mapping and biopsy. Currently, SLN mapping is routinely done in patients with many kinds of solid tumors to determine whether the cancer has spread beyond the primary tumor. However, the current protocol for SLN mapping, which involves the injection of an ionizing radiotracer, exposes patients and health-care professionals to ionizing radiation. In addition, in patients with head and neck cancer, the very complex structures in this region necessitate the use of high-resolution, three-dimensional imaging.

We have developed a dual-modality MR/optical imaging agent based on PG (13). We conjugated DTPA-Gd and NIR813 dye on the pendant carboxylic acid groups of PG to obtain PG-Gd-NIR813 (MW=60 kDa, 10% w/w Gd, 4.4% w/w NIR813). NIR813 fluoresces and absorbs at 813 nm. The conjugate, PG-Gd-NIR813, co-localized with isosulfan blue dye (the gold standard for SLN mapping), and its drainage into the SLN was confirmed by using optical imaging. When PG-Gd-NIR813 was injected intralingually in mice bearing DM 14 oral squamous cell carcinomas, MRI could detect not only the presence of PG-Gd-NIR813 but also the pattern of contrast agent distribution, which was different in metastatic and normal lymph nodes (Figure 6) (13). These results suggest that MRI could be used clinically to assess the presence of lymph node metastasis in patients with solid tumors. If MRI detected the presence of lymph node metastasis, surgery would have to be performed to remove the entire nodal basin; SLN biopsy and the associated waiting time would be unnecessary. If MRI did not detect lymph node metastasis, SLN mapping would be performed using preoperative and intraoperative NIR fluorescent imaging, without the use of an ionizing radiotracer or cumbersome interventional MRI. Because of its high detection sensitivity, NIR fluorescent imaging could also be used to monitor the success of complete resection of SLNs during surgery.

Figure 6.

Visualization of cervical lymph nodes after interstitial injection of PG-DTPA-Gd-NIR813 (0.02 mmol Gd/kg) into the tongue of a normal mouse (A–D) and a mouse with a human DM14 squamous cell carcinoma xenograft in the tongue (E–H). A and E, T1-weighted coronal image acquired 2 h after contrast injection. B and F, Overlay of white light and near-infrared fluorescence (NIRF) images 24 h after contrast injection. C and G, NIRF images of mice without skin. D and H, NIRF images of resected lymph nodes. The white arrows indicate sentinel lymph nodes, and red arrow indicates the primary tumor. I–K, Microphotographs of hematoxylin-eosin-stained sections of (I) normal lymph node, (J) metastatic lymph node, and (K) tumor-bearing tongue indicating the presence of micrometastases, presumably in-transit metastases in a lymphatic duct. Note the difference in the pattern of contrast distribution in the lymph nodes between normal and tumor-bearing mice in the magnetic resonance images (adapted from (13))

We also used PG-Gd-NIR813 to elucidate the mechanism behind the selective accumulation of PG in tumor necrotic regions. Rats bearing C6 tumors were intravenously injected with PG-Gd-NIR813 and subjected to in vivo imaging 48 h later. Figure 7A shows that PG-Gd-NIR813 was distributed toward the central zone of the tumor. The region of MRI signal enhancement correlated with the necrotic area in the tumor (Figure 7B). Using a fluorescence microscope, we found that PG-Gd-NIR813 co-localized with TUNEL-positive apoptotic cells but not with the viable tumor cells (Figure 7B). These results confirmed that PG-Gd-NIR813 targets the tumor apoptotic/necrotic area (93).

Figure 7.

(A) Representative MR imaging of C6 tumor-bearing nude rats, before and 48 h after i.v. injection of PG-Gd-NIR813 at a dose of 0.20 mmol of Gd/kg (50 nmol NIR813). Arrows, tumors. (B) Histological evaluation of tumor distribution of PG-Gd-NIR813 in C6-bearing nude rats after in vivo imaging. H&E staining shows necrotic regions in the tumors as light pink staining. Fluorescent micrographs of the adjacent tumor slide depicted the co-localization of PG-Gd-NIR813 with TUNEL-stained tumor cells. Cell nuclei were counterstained with DAPI. N, necrotic area; T, tumor. Bar, 50 μm. (adapted from (93))

To evaluate the role of macrophages in the recruitment of PG-Gd-NIR to necrotic areas, a macrophage depletion experiment was performed. In this experiment, BALB/c mice bearing A20 syngeneic tumors were treated with intravenous injection of clodronate-loaded liposomes to deplete circulating macrophages. Fluorescence molecular tomography (FMT) imaging showed that the mice treated with clodronate-loaded liposomes had significantly lower tumor uptake of PG-Gd-NIR813 than the control mice (Figure 8A). Quantitative analysis of relative fluorescence intensity revealed a decrease in PG-Gd-NIR813 signals of about sevenfold in tumors from mice with the macrophage-depleting treatment compared to those without treatment (Figure 8B). Immunohistochemical evaluation showed colocalization of PG-Gd-NIR813 and CD68⁺ macrophages in the tumor necrotic regions in the mice not treated with the macrophage-depleting agent. Twenty-four hours after macrophage depletion, both PG-Gd-NIR813 and CD68⁺ macrophages in tumor necrotic areas significantly decreased (Figure 8C, arrows). In tumor necrotic areas where CD68⁺ macrophages were not depleted, strong PG-Gd-NIR813 fluorescence signals could still be found (Figure 8C, arrowheads). Similar results were found in nude rats bearing C6 glioma tumors, in which complete loss of the fluorescent signal of PG-Gd-NIR813 in tumor necrotic areas was achieved after administration of the macrophage-depleting agent. Both CD68⁺ and CD169⁺ macrophages were completely depleted in the necrotic regions of A20 tumors in mice injected with liposomal clodronate.

Figure 8.

Intratumoral distribution of PG-Gd-NIR813 following macrophage depletion treatment. (A) In vivo fluorescence molecular tomography (FMT) imaging of PG-Gd-NIR813 in A20 tumor-bearing BALB/c mice with or without treatment with clodronate-loaded liposomes containing 1 mg of clodronate. Twenty-four hours after intravenous injection of the clodronate-loaded liposomes or saline, all the mice received 0.2 mmol/kg of PG-Gd-NIR813 and were imaged again 24 h later (n=5/group). (B) Fluorescent intensities of PG-Gd-NIR813 in A20 tumors from FMT imaging in mice injected with macrophage-depleting agent compared with saline control. (C) Fluorescence micrographs of A20 tumors in mice injected with PG-Gd-NIR813 (green) with or without macrophage depletion. The tumor-associated macrophages were stained with CD68 (red). The adjacent slices were stained with hematoxylin-eosin. N, necrosis. Arrows, tumor necrosis region with complete depletion of macrophages. Arrowheads, tumor necrotic area where CD68+ macrophages are partially depleted. Bar, 2 mm. (adapted from (93)).

Our current data indicate that PG-Gd-NIR813 was phagocytized by the M2 subset of tumor-infiltrating macrophages, which are recruited to the perinecrotic areas of tumors (93). The role of these infiltrating macrophages is likely multifaceted, involving both the removal of tumor debris and the promotion of angiogenesis. Similar mechanisms may have contributed to the accumulation of PG-TXL conjugate in tumor necrotic areas (86). As M2 macrophages in general promote tumor growth, selective retention of PG-TXL by M2 macrophages and subsequent intratumoral release of paclitaxel may be partially responsible for the significantly increased antitumor efficacy of PG-TXL used alone (63, 64) and in combination with radiotherapy as compared to the parent drug (70).

5 SUMMARY AND OUTLOOK

In summary, the efficacy of anticancer drugs and imaging agents can be improved by conjugating them to biomedical polymers, which modifies their pharmacokinetics and improves their tumor targeting efficiency. Molecular imaging provides an effective tool for noninvasive and continuous evaluation of in vivo delivery of polymer–drug conjugates in real time and can help elucidate the complex mechanisms for the improved efficacy of such conjugates. The combination of drug delivery, radiotherapy, and molecular imaging for treatment show a more efficient and promising regimen for eradicating the disease.