HPMA copolymer-cyclic RGD conjugates for tumor targeting

This review describes the design and development of N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-cyclic RGD conjugates for targeting tumor angiogenesis. Relative to non-targetable systems, HPMA copolymer-RGD4C and -RGDfK conjugates have shown increased tumor accumulation in a variety of solid tumors including prostate, lung, and breast tumor xenografts. Compared to free peptides, copolymers had increased tumor accumulation and decreased uptake in non-target organs such as the liver and spleen. Clinically relevant imaging agents such as ^99mTc, ¹¹¹In, and Gd enabled in vivo imaging of the constructs by scintigraphy and magnetic resonance techniques. Targeted delivery of ⁹⁰Y, a radiotherapeutic agent by HPMA copolymer-RGD4C conjugates resulted in tumor size reduction in mice bearing prostate tumor xenografts. Delivery of the geldanamycin derivative 17-(6-aminohexylamino)-17-demethoxygeldanamycin by HPMA copolymer-RGDfK conjugates resulted in increased tumor concentration of the free drug in a prostate xenograft model. These constructs show promise for targeted delivery of therapeutics and imaging agents to solid tumors.     

A novel αVβ3 ligand-modified HPMA copolymers for anticancer drug delivery

The integrin α_Vβ₃ receptor emerged as one of the most promising targets owing to its high expression on the surface of various malignant tumour cells and tumour angiogenesis endothelial cells, but with little expression in mature endothelial cells and the majority of normal cells. Here, we report a new targeting ligand FQSIYPpIK (FQS) with high affinity to integrin α_Vβ₃ receptor. To take the advantage of the particular interaction between FQS and integrin α_Vβ₃ receptor, FQS was linked to N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers. A model drug doxorubicin (DOX) was simultaneously conjugated to the same HPMA copolymers via pH-sensitive hydrazone linkages (FQS–HPMA–DOX). In in vitro study, FQS–HPMA–DOX could be internalised into α_Vβ₃ receptor-overexpressed B16F10 cells via a highly specific ligand???receptor pathway (5.0 times and 4.5 times higher cellular internalisation than HPMA–DOX and a scrambled peptide (s)-FQS (sequence: SYFIPKQIp)-modified copolymers ((s)-FQS–HPMA–DOX)). It is worth noting that compared with the classical α_Vβ₃ ligand cRGDfK-modified HPMA copolymers (cRGDfK–HPMA–DOX), FQS–HPMA–DOX also showed superior targeting efficiency. In in vivo study in the B16F10 melanoma bearing mice model showed the antitumour efficiency of FQS–HPMA–DOX (83.9%) were significantly higher than HPMA–DOX (44.9%) and cRGDfK–HPMA–DOX (77.5%). These results suggest that FQS peptide can act as an effective targeting ligand for the delivery of therapeutic agents.     

Spacer length impacts the efficacy of targeted docetaxel conjugates in prostate-specific membrane antigen expressing prostate cancer

Abstract

Combination of targeted delivery and controlled release is a powerful technique for cancer treatment. In this paper, we describe the design, synthesis, structure validation and biological properties of targeted and non-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel conjugates. Docetaxel (DTX) was conjugated to HPMA copolymer via a tetrapeptide spacer (–GFLG-). 3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) was used as the targeting moiety to actively deliver DTX for treatment of Prostate-Specific Membrane Antigen (PSMA) expressing prostate cancer. Short and long spacer DUPA monomers were prepared, and four HPMA copolymer – DTX conjugates (non-targeted, two targeted with short spacer of different molecular weight and targeted with long spacer) were prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) copolymerization. Following confirmation of PSMA expression on C4-2 cell line, the DTX conjugates’ in vitro cytotoxicity was tested against C4-2 tumor cells and their anticancer efficacies were assessed in nude mice bearing s.c. human prostate adenocarcinoma C4-2 xenografts. The in vivo results show that the spacer length between targeting moieties and HPMA copolymer backbone can significantly affect the treatment efficacy of DTX conjugates against C4-2 tumor bearing nu/nu mice. Moreover, histological analysis indicated that the DUPA-targeted DTX conjugate with longer spacer had no toxicity in major organs of treated mice.     

Targetable HPMA Copolymer–Aminohexylgeldanamycin Conjugates for Prostate Cancer Therapy

Purpose  This study focuses on the synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–cyclo-RGD (Arg-Gly-Asp) conjugates for delivery of geldanamycin to prostate tumors. Materials and Methods  HPMA copolymers containing aminohexylgeldanamycin (AH-GDM) with and without the targeting peptide RGDfK were synthesized and characterized. Drug release from copolymers was evaluated using cathepsin B. Competitive binding of copolymer conjugates to α_vβ₃ integrin was evaluated in prostate cancer (PC-3) and endothelial (HUVEC) cell lines and in vitro growth inhibition was assessed. The maximum tolerated dose for single i.v. injections of free drug and the conjugates was established in nude mice. Results  HPMA copolymers containing AH-GDM and RGDfK showed active binding to the α_vβ₃ integrin similar to that of free peptide. Similarly, growth inhibition of cells by conjugates was comparable to that of the free drug. Single intravenous doses of HPMA copolymer–AH-GDM–RGDfK conjugates in mice were tolerated at 80 mg/kg drug equivalent, while free drug caused morbidity at 40 mg/kg. No signs of toxicity were present in mice receiving HPMA copolymer-AH-GDM-RGDfK over the 14-day evaluation period. Conclusion  Results of in vitro activity and in vivo tolerability experiments hold promise for the utility of HPMA copolymer–AH-GDM–RGDfK conjugates for treatment of prostate cancer with greater efficacy and reduced toxicity.     

HPMA-copolymer conjugates targeted to tumor endothelium using synthetic oligopeptides

Synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA)-copolymer–based drug carriers targeted on specific receptors in the membrane of endothelial cells by oligopeptides (GRGDG, cyclo(RGDfK), and PHSCN) are described in this study. The copolymers containing targeting oligopeptides bound to the polymer via dodeca(ethylene glycol) spacer showed a receptor-specific time-dependent uptake with selected endothelial cell lines. The polymers were labeled with a fluorescent dye to enable monitoring of the interaction of the polymer conjugate with cells using fluorescence microscopy. Cellular uptake and apoptosis induction have been studied in vitro using various cell lines (EA.hy926, 3T3, SW620, and EL4). In vivo accumulation of the conjugate specifically targeted with cyclo(RGDfK) within the tumor vasculature was detected using fluorescence intravital microscopy in mice. The conjugate targeted by cyclo(RGDfK) was accumulated preferentially in the periphery of the growing tumor suggesting that the cyclo(RGDfK) peptide targets the polymer conjugate to the site of neoangiogenesis, rather than to the tumor mass.     

A combination of LightOn gene expression system and tumor microenvironment-responsive nanoparticle delivery system for targeted breast cancer therapy

A light-switchable transgene system called LightOn gene expression system could regulate gene expression with a high on/off ratio under blue light, and have great potential for spatiotemporally controllable gene expression. We developed a nanoparticle drug delivery system (NDDS) to achieve tumor microenvironment-responsive and targeted delivery of diphtheria toxin A (DTA) fragment-encoded plasmids to tumor sites. The expression of DTA was induced by exposure to blue light. Nanoparticles composed of polyethylenimine and vitamin E succinate linked by a disulfide bond, and PEGylated hyaluronic acid modified with RGD peptide, accumulated in tumor tissues and were actively internalized into 4T1 cells via dual targeting to CD44 and α_vβ₃ receptors. The LightOn gene expression system was able to control target protein expression through regulation of the intensity or duration of blue light exposure. In vitro studies showed that light-induced DTA expression reduced 4T1 cell viability and induced apoptosis. Furthermore, the LightOn gene expression system enabled spatiotemporal control of the expression of DTA in a mouse 4T1 tumor xenograft model, which resulted in excellent antitumor effects, reduced tumor angiogenesis, and no systemic toxicity. The combination of the LightOn gene expression system and NDDS may be an effective strategy for treatment of breast cancer.     

Overcoming chemotherapy resistance via simultaneous drug-efflux circumvention and mitochondrial targeting

Multidrug resistance (MDR) has been considered as a huge challenge to the effective chemotherapy. Therefore, it is necessary to develop new strategies to effectively overcome MDR. Here, based on the previous research of N-(2-hydroxypropyl)methacrylamide (HPMA) polymer–drug conjugates, we designed an effective system that combined drug-efflux circumvention and mitochondria targeting of anticancer drug doxorubicin (Dox). Briefly, Dox was modified with mitochondrial membrane penetrating peptide (MPP) and then attached to (HPMA) copolymers (P-M-Dox). Our study showed that macromolecular HPMA copolymers successfully bypassed drug efflux pumps and escorted Dox into resistant MCF-7/ADR cells via endocytic pathway. Subsequently, the mitochondria accumulation of drugs was significantly enhanced with 11.6-fold increase by MPP modification. The excellent mitochondria targeting then resulted in significant enhancement of reactive oxygen species (ROS) as well as reduction of adenosine triphosphate (ATP) production, which could further inhibit drug efflux and resistant cancer cell growth. By reversing Dox resistance, P-M-Dox achieved much better suppression in the growth of 3D MCF-7/ADR tumor spheroids compared with free Dox. Hence, our study provides a promising approach to treat drug-resistant cancer through simultaneous drug efflux circumvention and direct mitochondria delivery.     

<Emphasis Type="Italic">αvβ</Emphasis>3 Integrin Receptor Targeting and Near-Infrared Imaging of Solid Tumors Using Surface-Modified Nanoliposomes

Purpose

Abundance of receptors on tumor vasculature presents a prominent target for theranostic applications. The alphavbeta3 integrin receptors expressed on vascular endothelial cells during angiogenesis were therefore considered targets for imaging. Non-invasive visualization of tumor growth and/or delivery systems can appreciate tumor localization and disposition kinetics of carriers, respectively. Herein, we report near-infrared fluorescence imaging (NIRFI) of solid tumors using targeted fluorescence nanoliposomes in vivo.

Methods

Fluorescence nanoliposomes surface modified with cRGD-peptide were injected into CD1 athymic (nu/nu) mice bearing C6 glioblastoma xenografts (300 mm³). At different time points, mice were subjected to NIRFI for visualization of tumor xenografts and nanocarrier tracing in vivo.

Results

NIRFI showed tumor localization of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide (DiR₁₈)-incorporated-targeted liposomes with maximum tumor-to-tissue occurring at 24-h post-liposome administration. Interaction of integrin receptors with targeted liposomes had contributed to an intense NIRF signal. Molecular studies showed an elevated expression of alphavbeta3 integrin receptors in tumor xenografts.

Conclusion

From the studies, it can be concluded that non-invasive localization of tumors and tracing of liposome carriers had been achieved using receptor targeting and NIRFI approaches.

     

Synthesis and in vitro anti-tumor activity of novel HPMA copolymer-drug conjugates with potential cell surface targeting property for carcinoma cells

In several groups of malignant tumors including head and neck tumors, a protein named Hsp47/CBP2 leaked from the cell was expressed on the tumor cell surface. Several synthetic peptides have been identified as effective ligands for binding to Hsp47/CBP2. This study has focused on the synthesis and in vitro characterization of a targeting delivery system of 5-fluorouracil (5-FU) to human head and neck squamous cell carcinoma (HNSCC) in order to improve anti-cancer efficacy and reduce dose-limiting toxicity of 5-FU. An N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, with Hsp47/CBP2 binding peptide sequence (namely WHYPWFQNWAMA) as a targeting ligand, was synthesized by a novel and simplified synthetic route. Under the controlled synthetic conditions, 1,3-dimethylol-5-FU, derived from 5-FU, was attached to the HPMA copolymer backbone via the lysosomally degradable GFLG linker, while the WHYPWFQNWAMA was conjugated via a non-degradable Gly-Gly (GG) linker. A control polymer without targeting moiety was also synthesized (P-FU). The in vitro cytotoxicity, internalization and apoptosis assays of the polymeric conjugates were evaluated. The characteristic apoptotic morphological changes were also assessed. Compared to 5-FU and P-FU, the HPMA copolymer containing the Hsp47/CBP2 binding peptide (P-FU-peptide) exhibited the highest cytotoxic efficacy to cell line of human head and neck squamous cell carcinoma (p < 0.05) and was internalized much faster than P-FU, especially after being incubated for 30 min. Both of the morphology and apoptosis analyses demonstrated that the treatment of P-FU-peptide resulted in more apoptotic and necrotic induction of tumor cells than P-FU. Meanwhile, the rate of apoptosis induced by P-FU-peptide was higher than that of necrosis. In summary, the HPMA copolymer-Hsp47/CBP2 binding peptide conjugates showed a promising future for the treatment of HNSCC with improved efficacy.     

Active tumor targeting of nanomaterials using folic acid, transferrin and integrin receptors

Folic acid, transferrin and integrin alpha v beta 3 (αvβ3) receptors are overexpressed in various cancer cell lines. Ligands having high affinity for these receptors are often conjugated to nanocarriers to facilitate the tumor localization of therapeutic agents. In this review the use of these ligands for targeted delivery using liposomes, dendrimers and (N-(2-hydroxypropyl) methacrylamide) (HPMA) copolymers is discussed. Emphasis is placed on discussing drug delivery systems that have been optimized for in-vitro binding as well as in-vivo pharmacokinetics. Our aim is to understand the various factors influencing the targeting ability of nanocarriers.     

In Vitro and In Vivo Effect of HPMA Copolymer-bound Doxorubicin Targeted to Transferrin Receptor of B-cell Lymphoma 38C13

N -(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing the anticancer agent doxorubicin and targeted to the transferrin receptor either with anti-mouse CD71 monoclonal antibody (mAb) or with transferrin were synthesized to evaluate their binding and anti-proliferative activity in vitro and anti-tumor potential against 38C13 B-cell lymphoma in vivo. Both the doxorubicin and the targeting moieties were bound to HPMA copolymer chain by aminolysis via a Gly-Phe (d, l) -Leu-Gly spacer to ensure controlled intracellular release of the conjugated drug. We demonstrated that HPMA copolymer-bound doxorubicin targeted to the transferrin receptor with anti-mouse CD71 mAb strongly retards tumor growth, prolongs the survival and completely cures three out of nine experimental mice with established 38C13 tumors. The conjugate targeted with transferrin was less effective in vitro as well as in vivo. It completely cured only one out of seven experimental mice. Free or non-targeted HPMA copolymer-bound doxorubicin showed only a mild anti-tumor effect within the therapeutic schedule used. In vitro, HPMA copolymer-bound doxorubicin targeted with anti-mouse CD71 mAb shows approximately 4-fold higher cytotoxic effect than HPMA copolymer-bound doxorubicin targeted with transferin and 9-fold higher cytotoxic effect than non-targeted HPMA copolymer-bound doxorubicin.     

In vitro and in vivo effect of HPMA copolymer-bound doxorubicin targeted to transferrin receptor of B-cell lymphoma 38C13

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing the anticancer agent doxorubicin and targeted to the transferrin receptor either with anti-mouse CD71 monoclonal antibody (mAb) or with transferrin were synthesized to evaluate their binding and anti-proliferative activity in vitro and anti-tumor potential against 38C13 B-cell lymphoma in vivo. Both the doxorubicin and the targeting moieties were bound to HPMA copolymer chain by aminolysis via a Gly-Phe(D,L)-Leu-Gly spacer to ensure controlled intracellular release of the conjugated drug. We demonstrated that HPMA copolymer-bound doxorubicin targeted to the transferrin receptor with anti-mouse CD71 mAb strongly retards tumor growth, prolongs the survival and completely cures three out of nine experimental mice with established 38C13 tumors. The conjugate targeted with transferrin was less effective in vitro as well as in vivo. It completely cured only one out of seven experimental mice. Free or non-targeted HPMA copolymer-bound doxorubicin showed only a mild anti-tumor effect within the therapeutic schedule used. In vitro, HPMA copolymer-bound doxorubicin targeted with anti-mouse CD71 mAb shows approximately 4-fold higher cytotoxic effect than HPMA copolymer-bound doxorubicin targeted with transferin and 9-fold higher cytotoxic effect than non-targeted HPMA copolymer-bound doxorubicin.     

Noninvasive Visualization of Pharmacokinetics,Biodistribution and Tumor Targeting of Poly[<Emphasis Type="BoldItalic">N</Emphasis>-(2-hydroxypropyl)methacrylamide] in Mice Using Contrast Enhanced MRI

Purpose To study a non-invasive method of using contrast enhanced magnetic resonance imaging (MRI) to visualize the real-time pharmacokinetics, biodistribution and tumor accumulation of paramagnetically labeled poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) copolymer conjugates with different molecular weights and spacers in tumor-bearing mice. Materials and Methods Paramagnetically labeled HPMA copolymer conjugates were synthesized by free radical copolymerization of HPMA with monomers containing a chelating ligand, followed by complexation with Gd(OAc)₃. A stable paramagnetic chelate, Gd-DO3A, was conjugated to the copolymers via a degradable spacer GlyPheLeuGly and a non-degradable spacer GlyGly, respectively. The conjugates with molecular weights of 28, 60 and 121 kDa and narrow molecular weight distributions were prepared by fractionation with size exclusion chromatography. The conjugates were injected into athymic nude mice bearing MDA-MB-231 human breast carcinoma xenografts via a tail vein. MR images were acquired before and at various time points after the injection with a 3D FLASH sequence and a 2D spin-echo sequence at 3T. Pharmacokinetics, biodistribution and tumor accumulation of the conjugates were visualized based on the contrast enhancement in the blood, major organs and tumor tissue at various time points. The size effect of the conjugates was analyzed among the conjugates. Results Contrast enhanced MRI resulted in a real-time, three-dimensional visualization of blood circulation, pharmacokinetics, biodistribution and tumor accumulation of the conjugates, and the size effect on these pharmaceutical properties. HPMA copolymer conjugates with high molecular weight had a prolonged blood circulation time and high passive tumor targeting efficiency. Non-biodegradable HPMA copolymers with molecular weights higher than the threshold of renal filtration demonstrated higher efficiency for tumor drug delivery than biodegradable poly(L-glutamic acid). Conclusions Contrast enhanced MRI is an effective method for non-invasive visualization of in vivo properties of the paramagnetically labeled polymer conjugates in preclinical studies.     

Feasibility of Using a Bone-Targeted,Macromolecular Delivery System Coupled with Prostaglandin E<Subscript>1</Subscript> to Promote Bone Formation in Aged,Estrogen-Deficient Rats

Purpose  Macromolecular delivery systems have therapeutic uses because of their ability to deliver and release drugs to specific tissues. The uptake and localization of HPMA copolymers using Asp₈ as the bone-targeting moiety was determined in aged, ovariectomized (ovx) rats. PGE₁ was attached via a cathepsin K-sensitive linkage to HPMA copolymer–Asp₈ conjugate and was tested to determine if it could promote bone formation. Materials and Methods  The uptake of FITC-labeled HPMA copolymer–Asp₈ conjugate (P-Asp₈-FITC) on bone surfaces was compared with the mineralization marker, tetracycline. Then a targeted PGE₁-HPMA copolymer conjugate (P-Asp₈-FITC-PGE₁) was given as a single injection and its effects on bone formation were measured 4 weeks later. Results  P-Asp₈-FITC preferentially deposited on resorption surfaces, unlike tetracycline. A single injection of P-Asp₈-FITC-PGE₁ resulted in greater indices of bone formation in aged, ovx rats. Conclusions  HPMA copolymers can be targeted to bone surfaces using Asp₈, with preferential uptake on resorption surfaces. Additionally, PGE₁ attached to the Asp₈-targeted HPMA copolymers and given by a single injection resulted in greater bone formation measured 4 weeks later. This initial in vivo study suggests that macromolecular delivery systems targeted to bone may offer some therapeutic opportunities and advantages for the treatment of skeletal diseases.     

Increasing the antitumor efficacy of doxorubicin-loaded liposomes with peptides anchored via a chelator lipid

The therapeutic efficacy of anticancer drugs like doxorubicin can be significantly increased by their incorporation into liposomes, but an ability to actively target the drug-containing liposomes to tumors could well provide an even greater curative effect. In this work, a commercial preparation of doxorubicin-loaded liposomes (Caelyx) was modified by incorporation of the metal chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA₃-DTDA) to enable engraftment of histidine-tagged targeting molecules. Our results show that when engrafted with p15-RGR, a His-tagged peptide containing a sequence purported to bind platelet-derived growth factor receptor β (PDGFRβ), NTA₃-DTDA-containing Caelyx (3NTA-Caelyx) can be targeted to NIH-3T3 cells in vitro, leading to increased cytotoxicity compared with non-targeted 3NTA-Caelyx. PDGFRβ is known to be expressed on pericytes in the tumor vasculature; however, when radiolabeled p15-RGR liposomes were administered to mice bearing subcutaneous B16-F1 tumors, minimal accumulation into tumors was observed. In contrast, an alternative targeting peptide, p46-RGD, was found to actively direct liposomes to tumors (4.7 %ID/g). Importantly, when injected into tumor-bearing mice, p46-RGD-engrafted 3NTA-Caelyx significantly decreased the tumor growth rate compared with controls. These results indicate that the incorporation of NTA₃-DTDA into liposomal drugs could represent a simple modification to the drug to allow engraftment of targeting molecules and to increase its efficacy.     

Evaluation of a 188Re‐radiolabeled Arg‐Gly‐Asp peptide for tumor overexpressed αvβ3 receptors

To develop a peptide‐based radiopharmaceutical for the therapy of α_vβ₃ receptors overexpressed tumors, we have prepared a novel Arg‐Gly‐Asp (RGD) peptide (HCRGDCF(D)CRGDC, P12) radiolabeled with ¹⁸⁸Re. With His acid at the end of the peptide containing RGD, the label efficiency was more than 95% within 30 min. The peptide binds to human glioblastoma U87MG cells with high affinity [IC₅₀ = 86.3 nm]. The stability of ¹⁸⁸Re‐P12 in vitro was also investigated. More than 80% of radioactivity was kept in the peptide after 4 h incubation in phosphate buffer solution (pH = 7.4) or calf serum under physiological conditions. Biodistribution of this radiocompound was carried out in mice bearing S180 tumor. Fast clearance of ¹⁸⁸Re‐peptide from blood and specific uptakes by tumors realized higher tumor‐to‐blood ratio (1.80) 4 h post‐injection. Obvious difference was observed between the blocking and unblocking experiments in whole body autoradiography imaging. These results have demonstrated the potential of ¹⁸⁸Re‐labeling RGD as a radiotherapeutic agent.     

Enhanced Anti-Rheumatoid Arthritis Activity of Total Alkaloids from Picrasma Quassioides in Collagen-Induced Arthritis Rats by a Targeted Drug Delivery System

New drug delivery systems have rarely been used in the formulation of traditional Chinese medicine, especially those that are crude active Chinese medicinal ingredients. In the present study, hyaluronic acid decorated lipid-polymer hybrid nanoparticles were used to prepare a targeted drug delivery system (TDDS) for total alkaloid extract from Picrasma quassioides (TAPQ) to improve its targeting property and anti-inflammatory activity. Picrasma quassioides, a common-used traditional Chinese medicine (TCM), containing a series of hydrophobic total alkaloids including β-carboline and canthin-6-one alkaloids show great anti-inflammatory activity. However, its high toxicity (IC₅₀= 8.088±0.903 μg/ml), poor water solubility (need to dissolve with 0.8% Tween-80) and poor targeting property severely limits its clinical application. Herein, hyaluronic acid (HA) decorated lipid-polymer hybrid nanoparticles loaded with TAPQ (TAPQ-NPs) were designed to overcome above mentioned deficiencies. TAPQ-NPs have good water solubility, strong anti-inflammatory activity and great joint targeting property. The in vitro anti-inflammatory activity assay showed that the efficacy of TAPQ-NPs was significantly higher than TAPQ(P<0.001). Animal experiments showed that the nanoparticles had good joint targeting property and had strong inhibitory activity against collagen-induced arthritis (CIA). These results indicate that the application of this novel targeted drug delivery system in the formulation of traditional Chinese medicine is feasible.     

Improving the efficacy of combined modality anticancer therapy using HPMA copolymer-based nanomedicine formulations

Copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) are prototypic and well-characterized polymeric drug carriers that have been broadly implemented in the delivery of anticancer agents. HPMA copolymers circulate for prolonged periods of time, and by means of the Enhanced Permeability and Retention (EPR) effect, they localize to tumors both effectively and selectively. Because of their beneficial biodistribution, and because of the fact that they are able to improve the balance between the efficacy and the toxicity of chemotherapy, it is reasonable to assume that HPMA copolymers combine well with other treatment modalities. In the present review, efforts in this regard are summarized, and HPMA copolymers are shown to be able to beneficially interact with surgery, with radiotherapy, with hyperthermia, with photodynamic therapy, with chemotherapy and with each other. Together, the insights provided and the evidence obtained strongly suggest that HPMA copolymer-based nanomedicine formulations hold significant potential for improving the efficacy of combined modality anticancer therapy.     

Do HPMA copolymer conjugates have a future as clinically useful nanomedicines? A critical overview of current status and future opportunities

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing doxorubicin designed in the late 1970s/early 1980s as anticancer polymer therapeutics were the first synthetic polymer-based anticancer conjugates to enter clinical trial beginning in 1994. Early clinical results were promising, confirming activity in chemotherapy refractory patients and the safety of HPMA copolymers as a new polymer platform in this setting. Subsequent Phase I/II trials have investigated conjugates containing paclitaxel (PNU 166945), camptothecin (PNU 166148) (both failed in clinical trials underlining the importance of rational design), and most recently HPMA-copolymer platinates (AP5280 and then AP5346-ProLindac^TM) entered Phase II clinical development. There are a growing array of second generation HPMA copolymer-based systems involving combination therapy, incorporating putative targeting ligands, having an ever more complex architecture, and both drug and protein conjugates are being proposed as novel treatments for diseases other than cancer. Despite their promise, and the success of polymeric drugs and polymer-protein conjugates, no polymer-drug conjugate (HPMA copolymer-based or otherwise) has yet entered routine clinical use. It is timely to reflect on the progress made over the last 30 years, the relative merits of HPMA copolymers as a platform compared to other polymeric carriers, and comment on their future potential as polymer-based nanomedicines into the 21st century in comparison with the many alternative strategies now emerging for creation of nanopharmaceuticals.     

Noninvasive Monitoring of HPMA Copolymer–RGDfK Conjugates by Magnetic Resonance Imaging

Purpose  To evaluate the tumor targeting potential of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–gadolinium(Gd)–RGDfK conjugates by magnetic resonance (MR) T1-mapping. Methods  HPMA copolymers with and without RGDfK were synthesized to incorporate side chains for Gd chelation. The conjugates were characterized by their side-chain contents and r₁ relaxivity. In vitro integrin-binding affinities of polymeric conjugates were assessed via competitive cell binding assays on HUVEC endothelial cells and MDA-MB-231 breast cancer cells. In vivo MR imaging was performed on MDA-MB-231 tumor-bearing SCID mice at different time points using non-targetable and targetable polymers. The specificity of αvβ3 targeting was assessed by using non-paramagnetic targetable polymer to block αvβ3 integrins followed by injection of paramagnetic targetable polymers after 2 h. Results  The polymer conjugates showed relaxivities higher than Gd-DOTA. Endothelial cell binding studies showed that IC₅₀ values for the copolymer with RGDfK binding to αvβ3 integrin-positive HUVEC and MDA-MB-231 cells were similar to that of free peptide. Significantly lower T1 values were observed at the tumor site after 2 h using targetable conjugate (p < 0.012). In vivo blocking study showed significantly higher T1 values (p < 0.045) compared to targetable conjugate. Conclusion  These results demonstrate the potential of this conjugate as an effective targetable MR contrast agent for tumor imaging and therapy monitoring.