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Supplementary Figure S1 shows increased fluorescence image of S180 tumor treated with of HPMA-copolymer-conjugated zinc protoporphyrin (PZP) in combination with NO donors.

Enhanced permeability and retention (EPR) effect–based nanomedicine is a promising strategy for successful anticancer therapy. The EPR effect is based on tumor blood flow. Because advanced large tumors, as frequently seen in clinical settings, are heterogeneous, with regions of defective vasculature and blood flow, achieving the desired tumor drug delivery is difficult. Here, we utilized the EPR effect to increase drug delivery. To augment the EPR effect for improved therapeutic effects of nanomedicine, we exploited vascular mediators—the nitric oxide (NO) generators nitroglycerin (NG), hydroxyurea, and l-arginine. These compounds generate NO in tumors with relatively high selectivity. Using different nanosized drugs in our protocol significantly increased (1.5–2 times) delivery of nanomedicines to different solid tumor models, along with markedly improving (2–3-fold) the antitumor effects of these drugs. Also, in 7,12-dimethylbenz[a]anthracene–induced advanced end-stage breast cancer, often seen in clinical settings, 2 mg/kg polymer-conjugated pirarubicin (P-THP) with NG (0.2 mg/mouse) showed better effects than did 5 mg/kg P-THP, and 5 mg/kg P-THP used with NG resulted in cures or stable tumors (no tumor growth) for up to 120 days. Moreover, in a murine autochthonous azoxymethane/dextran sulfate sodium-induced colon cancer model, NO donors markedly improved the therapeutic effects of P-THP even after just one injection, results that were comparable with those achieved with three weekly P-THP treatments. These findings strongly suggest the potential usefulness of NO donors as EPR effect enhancers to improve the therapeutic efficacy of nanomedicines.

In this study, we compared the enhanced permeability and retention (EPR) effect, toxicity, and therapeutic effect of the conjugate of the linear polymer poly( N -(2-hydroxypropyl)methacrylamide) (HPMA) with pirarubicin with an M w below the renal threshold (39 g/mol) (named LINEAR) and the disulfide-linked tandem-polymeric dimer of the poly(HPMA)-pirarubicin conjugate with an M w above the renal threshold (93 g/mol) (named DIBLOCK). The DIBLOCK conjugate, which was susceptible to reductive degradation, showed both a better EPR effect (tumor delivery) (2.5 times greater at 24 h) and a prolonged plasma half-life. In addition, DIBLOCK had a better antitumor effect, as judged by percent survival, than did LINEAR (80% vs 65% at 150 days), without any apparent toxicity in an S180 tumor model. However, the LD 50 value of LINEAR was slightly higher than that of DIBLOCK (50 mg/kg vs 37.5 mg/kg, respectively). DIBLOCK required a longer time than LINEAR to reach maximum accumulation in the tumor. DIBLOCK also showed a greater time-dependent increase in the concentration in the tumor compared with the plasma concentration.

The aim of this study was to evaluate the use of cationic–hydrophilic copolymers for self-assembly with antisense oligonucleotides targeted to the bcl-2 mRNA in order to improve their biocompatibility and modulation of their pharmacokinetics for greater therapeutic usefulness. Examination of the ability of poly(trimethylammonioethyl methacrylate chloride)–poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA-b-pTMAEM) block copolymers to condense the oligonucleotide by fluorescence and electrophoresis techniques showed that complexes were formed more efficiently than with copolymers containing poly(ethylene glycol) blocks grafted onto the backbone of poly(l-lysine) (pLL-g-pEG). In addition, the copolymer pTMAEM-b-pHPMA produced oligonucleotide complexes with the most favourable physicochemical properties appropriate for in vivo applications. The complexes were small (approximately 36 nm in diameter), with low surface charge as measured by zeta potential, relatively stable to physiological salt conditions and could be formed at a DNA concentration of 500 μg/ml. Complex formation with the copolymer pTMAEM-b-pHPMA or pLL-g-pEG reduced the urinary clearance of the oligonucleotide after intravenous injection into mice. However after 30 min, the oligonucleotide complexes were cleared from the bloodstream. These results indicate that for the systemic delivery of oligonucleotides the polymer-derived complexes are not stable enough for prolonged circulation. Instead, these complexes may be more suitable for localised in vivo applications.

Binding of serum proteins to polyelectrolyte gene delivery complexes is thought to be an important factor limiting bloodstream circulation and restricting access to target tissues. Protein binding can also inhibit transfection activity in vitro. In this study a multivalent reactive hydrophilic polymer has been used to inhibit protein binding. This polymer is based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) bearing pendent oligopeptide (Gly-Phe-Leu-Gly) side chains terminated in reactive 4-nitrophenoxy groups (8.6 mol%). The polymer reacts with the primary amino groups of poly(L-lysine) (pLL) and produces a hydrophilic coating on the surface of pLL.DNA complexes (as measured by fluorescamine). The resulting pHPMA-coated complexes show a decreased surface charge (from +14 mV for pLL.DNA complexes to -25 mV for pHPMA-modified complexes) as measured by zeta potential analysis. The pHPMA-coated complexes also show a slightly increased average diameter (approximately 90 nm compared with 60 nm for pLL. DNA complexes) as viewed by atomic force and transmission electron microscopy and around 100 nm as viewed by photon correlation spectroscopy. They are completely resistant to protein interaction, as determined by turbidometry and SDS-polyacrylamide gel electrophoresis analysis of complexes isolated from plasma, and show significantly decreased nonspecific uptake into cells in vitro. Spare reactive ester groups can be used to conjugate targeting ligands (e.g. transferrin) on to the surface of the complex to provide a means of tissue-specific targeting and transfection. The properties of these complexes therefore make them promising candidates for targeted gene delivery, both in vitro and potentially in vivo.

Multidrug resistance (MDR) contributes to failure of chemotherapy. We here show that biodegradable polymeric nanogels are able to overcome MDR via folic acid targeting. The nanogels are based on hydroxyethyl methacrylamide-oligoglycolates-derivatized poly(hydroxyethyl methacrylamide-co-N-(2-azidoethyl)methacrylamide) (p(HEMAm-co-AzEMAm)-Gly-HEMAm), covalently loaded with the chemotherapeutic drug doxorubicin (DOX) and subsequently decorated with a folic acid-PEG conjugate via copper-free click chemistry. pH-Responsive drug release is achieved via the acid-labile hydrazone bond between DOX and the methacrylamide polymeric network. Cellular uptake and cytotoxicity analyses in folate receptor-positive B16F10 melanoma versus folate receptor-negative A549 lung carcinoma cells confirmed specific uptake of the targeted nanogels. Confocal microscopy demonstrated efficient internalization, lysosomal trafficking, drug release and nuclear localization of DOX. We also show that DOX resistance in 4T1 breast cancer cells results in upregulation of the folate receptor, and that folic acid targeted nanogels can be employed to bypass drug efflux pumps, resulting in highly efficient killing of resistant cancer cells. In conclusion, folic acid functionalized nanogels with pH-controlled drug release seem to hold significant potential for treating multidrug resistant malignancies.

Many conjugates of water-soluble polymers with biologically active molecules were developed during the last two decades. Although, therapeutic effects of these conjugates are affected by the properties of carriers, the properties of the attached drugs appear more important than the same carrier polymer in this case. Pirarubicin (THP), a tetrahydropyranyl derivative of doxorubicin (DOX), demonstrated more rapid cellular internalization and potent cytotoxicity than DOX. Here, we conjugated the THP or DOX to N-(2-hydroxypropyl)methacrylamide copolymer via a hydrazone bond. The polymeric prodrug conjugates, P-THP and P-DOX, respectively, had comparable hydrodynamic sizes and drug loading. Compared with P-DOX, P-THP showed approximately 10 times greater cellular uptake during a 240 min incubation and a cytotoxicity that was more than 10 times higher during a 72-h incubation. A marginal difference was seen in P-THP and P-DOX accumulation in the liver and kidney at 6 h after drug administration, but no significant difference occurred in the tumor drug concentration during 6-24 h after drug administration. Antitumor activity against xenograft human pancreatic tumor (SUIT2) in mice was greater for P-THP than for P-DOX. To sum up, the present study compared the biological behavior of two different drugs, each attached to an N-(2-hydroxypropyl)methacrylamide copolymer carrier, with regard to their uptake by tumor cells, body distribution, accumulation in tumors, cytotoxicity, and antitumor activity in vitro and in vivo. No differences in the tumor cell uptake of the polymer-drug conjugates, P-THP and P-DOX, were observed. In contrast, the intracellular uptake of free THP liberated from the P-THP was 25-30 times higher than that of DOX liberated from P-DOX. This finding indicates that proper selection of the carrier, and especially conjugated active pharmaceutical ingredient (API) are most critical for anticancer activity of the polymer-drug conjugates. THP, in this respect, was found to be a more preferable API for polymer conjugation than DOX. Hence the treatment based on enhanced permeability and retention (EPR) effect that targets more selectively to solid tumors can be best achieved with THP, although both polymer conjugates of DOX and THP exhibited the EPR effects and drug release profiles in acidic pH similarly.

Here, we present the synthesis, physicochemical, and preliminary biological characterization of micellar polymer-betulinic acid (BA) conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carriers, enabling the controlled release of cytotoxic BA derivatives in solid tumors or tumor cells. Various HPMA copolymer conjugates differing in the structure of the spacer between the drug and the carrier were synthesized, all designed for pH-triggered drug release in tumor tissue or tumor cells. The high molecular weight of the micellar conjugates should improve the uptake of the drug in solid tumors due to the Enhanced permeability and retention (EPR) effect. Nevertheless, only the conjugate containing BA with methylated carboxyl groups enabled pH-dependent controlled release in vitro. Moreover, drug release led to the disassembly of the micellar structure, which facilitated elimination of the water-soluble HPMA copolymer carrier from the body by renal filtration. The methylated BA derivative and its polymer conjugate exhibited high cytostatic activity against DLD-1, HT-29, and HeLa carcinoma cell lines and enhanced tumor accumulation in HT-29 xenograft in mice.

Water-soluble N -(2-hydroxypropyl)methacrylamide copolymer conjugates bearing the anticancer drugs doxorubicin (Dox) or pirarubicin (THP), P-gp inhibitors derived from reversin 121 (REV) or ritonavir (RIT)), or both anticancer drug and P-gp inhibitor were designed and synthesized. All biologically active molecules were attached to the polymer carrier via pH-sensitive spacer enabling controlled release in mild acidic environment modeling endosomes and lysosomes of tumor cells. The cytotoxicity of the conjugates against three sensitive and Dox-resistant neuroblastoma (NB) cell lines, applied alone or in combination, was studied in vitro . All conjugates containing THP displayed higher cytotoxicity against all three Dox-resistant NB cell lines compared with the corresponding Dox-containing conjugates. Furthermore, the cytotoxicity of conjugates containing both drug and P-gp inhibitor was up to 10 times higher than that of the conjugate containing only drug. In general, the polymer-drug conjugates showed higher cytotoxicity when conjugates containing inhibitors were added 8 or 16 h prior to treatment compared with conjugates bearing both the inhibitor and the drug. The difference in cytotoxicity was more pronounced at the 16-h time point. Moreover, higher inhibitor:drug ratios resulted in higher cytotoxicity. The cytotoxicity of the polymer-drug used in combination with polymer P-gp inhibitor was up to 84 times higher than that of the polymer-drug alone.

Targeted delivery combined with controlled drug release has a pivotal role in the future of personalized medicine. This review covers the principles, advantages, and drawbacks of passive and active targeting based on various polymer and magnetic iron oxide nanoparticle carriers with drug attached by both covalent and noncovalent pathways. Attention is devoted to the tailored conjugation of targeting ligands (e.g., enzymes, antibodies, peptides) to drug carrier systems. Similarly, the approaches toward controlled drug release are discussed. Various polymer-drug conjugates based, for example, on polyethylene glycol (PEG), N-(2-hydroxypropyl)methacrylamide (HPMA), polymeric micelles, and nanoparticle carriers are explored with respect to absorption, distribution, metabolism, and excretion (ADME scheme) of administrated drug. Design and structure of superparamagnetic iron oxide nanoparticles (SPION) and condensed magnetic clusters are classified according to the mechanism of noncovalent drug loading involving hydrophobic and electrostatic interactions, coordination chemistry, and encapsulation in porous materials. Principles of covalent conjugation of drugs with SPIONs including thermo- and pH-degradable bonds, amide linkage, redox-cleavable bonds, and enzymatically-cleavable bonds are also thoroughly described. Finally, results of clinical trials obtained with polymeric and magnetic carriers are analyzed highlighting the potential advantages and future directions in targeted anticancer therapy.

Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic behavior (∼300 nm submicrometer particles, ∼10 nm micelles and ∼4 nm flexible random coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions resulted in a nearly 10-fold reduction in systemic cytokines (toxicity). Moreover, both lymph node cytokine production and the magnitude of CD8 T cells induced against protein antigen increased with increasing polymer-TLR-7/8a hydrodynamic radius, with the submicrometer particle inducing the highest magnitude responses. Notably, CD8 T cell responses induced by polymer-TLR-7/8a were dependent on CCR2+ monocytes and IL-12, whereas responses by a small molecule TLR-7/8a that unexpectedly persisted in vaccine-site draining lymph nodes (T1/2 = 15 h) had less dependence on monocytes and IL-12 but required Type I IFNs. This study shows how modular properties of synthetic adjuvants can be chemically programmed to alter immunity in vivo through distinct immunological mechanisms.

Enhanced permeability and retention (EPR) effect–based nanomedicine is a promising strategy for successful anticancer therapy. The EPR effect is based on tumor blood flow. Because advanced large tumors, as frequently seen in clinical settings, are heterogeneous, with regions of defective vasculature and blood flow, achieving the desired tumor drug delivery is difficult. Here, we utilized the EPR effect to increase drug delivery. To augment the EPR effect for improved therapeutic effects of nanomedicine, we exploited vascular mediators—the nitric oxide (NO) generators nitroglycerin (NG), hydroxyurea, and l-arginine. These compounds generate NO in tumors with relatively high selectivity. Using different nanosized drugs in our protocol significantly increased (1.5–2 times) delivery of nanomedicines to different solid tumor models, along with markedly improving (2–3-fold) the antitumor effects of these drugs. Also, in 7,12-dimethylbenz[a]anthracene–induced advanced end-stage breast cancer, often seen in clinical settings, 2 mg/kg polymer-conjugated pirarubicin (P-THP) with NG (0.2 mg/mouse) showed better effects than did 5 mg/kg P-THP, and 5 mg/kg P-THP used with NG resulted in cures or stable tumors (no tumor growth) for up to 120 days. Moreover, in a murine autochthonous azoxymethane/dextran sulfate sodium-induced colon cancer model, NO donors markedly improved the therapeutic effects of P-THP even after just one injection, results that were comparable with those achieved with three weekly P-THP treatments. These findings strongly suggest the potential usefulness of NO donors as EPR effect enhancers to improve the therapeutic efficacy of nanomedicines.

Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named “iBodies”, consist of an HPMA copolymer decorated with low‐molecular‐weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live‐cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand.

A new drug-delivery system of polymer nanoparticles (NPs) bearing pinacol-type boronic ester and alkyne moieties displaying triggered self-immolative polymer degradation in the presence of reactive oxygen species (ROS) with the capability of cellular imaging is presented. The NPs specifically release their drug cargo under concentrations of ROS that are commonly found in the intracellular environment of certain tumors and of inflamed tissues and exhibit significant cytotoxicity to cancer cells compared to their non-ROS-responsive counterparts.

Interleukin-2 (IL-2) possesses a strong stimulatory activity for activated T and NK cells and it is an attractive molecule for immunotherapy. Nevertheless, extremely short half-life and severe toxicities associated with high-dose IL-2 treatment are serious and limiting drawbacks. In order to increase IL-2 half-life in vivo, we covalently conjugated synthetic semitelechelic polymeric carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) to IL-2. Thus, we synthesized IL-2-poly(HPMA) conjugate containing 2-3 polymer chains per IL-2 molecule in average. Such conjugate has lower biologic activity in comparison to IL-2 in vitro. However, it exerts much higher activity than IL-2 in vivo as shown by expansion of memory CD8+ T, NK, NKT, γδT and Treg cells. Moreover, IL-2-poly(HPMA) extremely effectively potentiates CD8+ T cell peptide-based vaccination. IL-2-poly(HPMA) shows also much longer half-time in circulation than IL-2 (-4 h versus -5 min). Collectively, modification of IL-2 with poly(HPMA) chains dramatically improves its potency and pharmacologic features in vivo, which have implications for immunotherapy. To our knowledge, this is the first proof-of-concept report of the use of polymer/protein modification of IL-2 to obtain more pronounced biological activity.

An effective chemotherapy for neoplastic diseases requires the use of drugs that can reach the site of action at a therapeutically efficacious concentration and maintain it at a constant level over a sufficient period of time with minimal side effects. Currently, conjugates of high-molecular-weight hydrophilic polymers or biocompatible nanoparticles with stimuli-releasable anticancer drugs are considered to be some of the most promising systems capable of fulfilling these criteria. In this work, conjugates of thermoresponsive diblock copolymers with the covalently bound cancerostatic drug pirarubicin (PIR) were synthesized as a reversible micelle-forming drug delivery system combining the benefits of the above-mentioned carriers. The diblock copolymer carriers were composed of hydrophilic poly[N-(2-hydroxypropyl)methacrylamide]-based block containing a small amount (∼ 5 mol %) of comonomer units with reactive hydrazide groups and a thermoresponsive poly[2-(2-methoxyethoxy)ethyl methacrylate] block. PIR was attached to the hydrophilic block of the copolymer through the pH-sensitive hydrazone bond designed to be stable in the bloodstream at pH 7.4 but to be degraded in an intratumoral/intracellular environment at pH 5-6. The temperature-induced conformation change of the thermoresponsive block (coil-globule transition), followed by self-assembly of the copolymer into a micellar structure, was controlled by the thermoresponsive block length and PIR content. The cytotoxicity and intracellular transport of the conjugates as well as the release of PIR from the conjugates inside the cells, followed by its accumulation in the cell nuclei, were evaluated in vitro using human colon adenocarcinoma (DLD-1) cell lines. It was demonstrated that the studied conjugates have a great potential to become efficacious in vivo pharmaceuticals.

High-molecular-weight star polymer drug nanocarriers intended for the treatment and/or visualisation of solid tumours were synthesised, and their physico-chemical and preliminary in vitro biological properties were determined. The water-soluble star polymer carriers were prepared by the grafting of poly(amido amine) (PAMAM) dendrimers by hetero-telechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, synthesised by the controlled radical Reversible Addition Fragmentation chain Transfer (RAFT) polymerisation. The well-defined star copolymers with Mw values ranging from 2 · 10(5) to 6 · 10(5) showing a low dispersity (approximately 1.2) were prepared in a high yield. A model anticancer drug, doxorubicin, was bound to the star polymer through a hydrazone bond, enabling the pH-controlled drug release in the target tumour tissue. The activated polymer arm ends of the star copolymer carrier enable a one-point attachment for the targeting ligands and/or a labelling moiety. In this study, the model TAMRA fluorescent dye was used to prove the feasibility of the polymer carrier visualisation by optical imaging in vitro. The tailor-made structure of the star polymer carriers should facilitate the synthesis of targeted polymer-drug conjugates, even polymer theranostics, for simultaneous tumour drug delivery and imaging.

The potential use of polyester polymeric nanoparticles (NPs) as drug nanocarriers is well-documented. Nevertheless, structural changes due to hydrophobic drug loading and release have been rarely explored. Herein, we have used static and dynamic light scattering (SDLS), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM) and cryo-TEM to probe how the entrapment of a hydrophobic drug molecule changes the nanoparticles feature. The presence of the hydrophobic drug molecule modifies the inner structure of the NPs. The polymeric assemblies are characterized by differences in their densities (∼ 0.06 g cm-3 for poly(D,L-lactide) – PLA or poly(D,L-lactide-co-glycolide – PLGA) and 0.46 g cm-3 for poly[(butylene succinate)-co-(butylene dilinoleate)] − PBSBDL). They are thus water swollen in the drug-free condition. The NPs were further prepared by using the same polyesters and given amounts of the poorly water-soluble drug paclitaxel (PTX). The density (dNP), RG (radius of gyration), RH (hydrodynamic radius), RG/RH and R (contrast radius) have been monitored as a function of the amount of drug loaded. The drug entrapment increased the size of PLA and PLGA NPs. On the other hand, it also promoted the shrinkage of PBSBDL NPs. These observations revealed that changes in the inner structure of soft nanoparticles caused by drug loading is not straightforward and it mainly depends on the strength of van der Waals interactions between the polyester core and the probe which is connected to their chemical composition and hydrophobicity. These findings are crucial to understand the key physicochemical parameters involved in the interactions between drug and polymer that affects the final particle structure and influence its loading, release and degradation.

Here we present the polymer conjugates where the core formed by poly(amido amine) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing docetaxel (DTX) attached by a pH-sensitive hydrazone bond. DTX was derivatized with three different keto acids prior to attachment to the polymer carrier to introduce reactive keto groups into the drug. The therapeutic efficacy of such high-molecular-weight star conjugates is based on: (a) the enhanced permeability and retention (EPR) effect facilitating selective accumulation within solid tumors; (b) pH-controlled release of the drug, thus ensuring faster DTX release in the mildly acidic tumor microenvironment. The star DTX conjugate had a remarkably higher maximum tolerated dose in comparison with free DTX when administered as a single i.v. injection (∼160 mg kg−1vs. 40 mg kg−1 of DTX) in C57BL/6 mice. The star DTX conjugate showed significantly higher antitumor activity than free drug in the EL4 T cell lymphoma growing in syngeneic C57BL/6 mice even when given at the same dose (20 mg kg−1 of DTX eq.). Thus, the star DTX conjugates exert a much higher therapeutic activity and yet a lower systemic toxicity than free DTX.

Over the past few decades, numerous polymer drug carrier systems are designed and synthesized, and their properties are evaluated. Many of these systems are based on water-soluble polymer carriers of low-molecular-weight drugs and compounds, e.g., cytostatic agents, anti-inflammatory drugs, or multidrug resistance inhibitors, all covalently bound to a carrier by a biodegradable spacer that enables controlled release of the active molecule to achieve the desired pharmacological effect. Among others, the synthetic polymer carriers based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers are some of the most promising carriers for this purpose. This review focuses on advances in the development of HPMA copolymer carriers and their conjugates with anticancer drugs, with triggered drug activation in tumor tissue and especially in tumor cells. Specifically, this review highlights the improvements in polymer drug carrier design with respect to the structure of a spacer to influence controlled drug release and activation, and its impact on the drug pharmacokinetics, enhanced tumor uptake, cellular trafficking, and in vivo antitumor activity.

The effects of novel polymeric therapeutics based on water-soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bearing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters, or both, on the viability and the proliferation of the murine monocytic leukemia cell line P388 (parental cell line) and its doxorubicin-resistant subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone bond, and the ability of these conjugates to block the ATP driven P-glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121 showed a dose-dependent increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24 μM, and achieved an approximately 50-fold increase in sensitization at 24 μM. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-RIT showed moderate activity at 6 μM (∼10 times higher sensitization) and increased sensitization by 50-fold at 12 μM. The cytostatic activity of the P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-N═Dox toward the P388/MDR cells in vitro. A similar result was observed for P-Ahx-NH-N═MeOHe-RIT(Dox), which exhibited almost 10 times higher cytostatic activity than P-Ahx-NH-N═Dox.

In this study, we describe the synthesis, physico-chemical characterisation and results of the in vitro and in vivo evaluation of the biological behaviour of N-(2-hydroxypropyl)methacrylamide-based (HPMA) copolymer conjugates bearing doxorubicin (DOX) partly bound via a pH-sensitive hydrazone and partly via enzymatically degradable amide bonds, each contributing to a different anti-tumour mechanism of action of the polymer-doxorubicin conjugate. The following two types of HPMA copolymer drug carriers designed for passive tumour targeting were synthesised and compared: the linear non-degradable copolymer and the biodegradable high-molecular-weight (HMW) diblock copolymer. The HMW diblock copolymer carrier containing a degradable disulphide bond between the polymer blocks showed a rapid degradation in a buffer containing glutathione within the first few hours of incubation. In contrast to the conjugate with the amide bond-bound DOX requiring the presence of lysosomal enzymes to release DOX, the polymer-drug conjugate with the DOX bound via a hydrazone bond released DOX by pH-sensitive hydrolysis, which was significantly faster in a buffer of pH 5.0 (intracellular pH) than pH 7.4, mimicking the conditions in the bloodstream. The significant and comparable in vivo anti-tumour activity of the diblock HMW conjugate and an equimolar mixture of the conjugates differing in the DOX attachment method along with the development of cancer resistance during treatment with these conjugates demonstrated the high potential of these compounds in the development of new nanomedicines suitable for the treatment of solid tumours.

N-(2-Hydroxypropyl)methacrylamide copolymer containing hydrazide groups (PHPMA) conjugated with pirarubicin (THP) via a hydrazone bond (PHPMA-hyd-THP) is a drug conjugate that releases THP in the acidic milieu of a tumor. PHPMA-hyd-THP has an apparent Mw of 40,000 and a hydrodynamic diameter of 8.2±1.7nm but no apparent plasma protein binding. PHPMA-hyd-THP possesses two mechanisms of selectivity toward solid tumors and has potent antitumor action. The first one is drug accumulation in tumors that depends on the enhanced permeability and retention (EPR) effect, which results in a 4-20 times higher concentration of drug in the tumor than in normal tissues such as the heart, lung, and intestine. This accumulation in tumor tissue is in great contrast to that of conventional low-Mw THP. The second one is pH-dependent release of drug from PHPMA-hyd-THP: this conjugate released free THP more efficiently at a lower pH, which exists in tumors, and exerts cytotoxic activity. Free THP is known for its much faster uptake into tumor cells compared with doxorubicin. Thus, in our in vitro study, PHPMA-hyd-THP showed a higher cytotoxicity at the lower pH of tumor tissue than at the neutral pH of normal tissue. Furthermore, much more THP was liberated from the conjugate in acidic tumor tissue than in normal tissue. The EPR effect-dependent accumulation of PHPMA-hyd-THP and tumor-selective THP release in the tumor tissues led to highly tumor-selective drug accumulation, which continued for more than 72h, whereas the lowest free drug concentration was detected in normal tissues at 24h and no longer at a later time. In conclusion, we determined in our study here that the acid-cleavable PHPMA-hyd-THP conjugate had an excellent antitumor effect without appreciable adverse effects.

Various click chemistry azide–alkyne cycloaddition reactions were used to attach azide group-terminated peptides to polymer drug carriers in an effort to conjugate biologically active molecules with polymer drug carriers by directly binding unprotected peptides to these polymers. Three methods using click chemistry to conjugate the azide group-containing molecules with synthetic polymers were compared: (1) click chemistry with a Cu(I) catalyst in aqueous and organic solvents, (2) click reactions using ruthenium complex catalysts in DMF and (3) metal-free click chemistry based on a dibenzocyclooctyne (DBCO) reactive group. The suitability of these reactions was verified for the non-covalent attachment of targeting moieties to these polymer carriers via peptide–peptide interactions. Moreover, RAFT polymerization was suggested for the synthesis of semitelechelic copolymers containing a single DBCO group at the polymer chain end and for the preparation of well-defined diblock copolymer drug carriers consisting of specific peptide and hydrophilic polymer blocks.

In recent years, polymer drug carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with pH-triggered drug release have shown enhanced uptake in solid tumors and excellent antitumor activity. Here, the impact of the structure of the acid-labile spacer between the drug and the polymer carrier on the biodistribution of both the drug and the carrier was studied using in vivo noninvasive multispectral optical imaging of dual fluorescently labeled HPMA copolymers. Five different spacers containing a pH-sensitive hydrazone bond were synthesized and used to combine a fluorescent model drug with a polymer backbone, conjugated with another non-releasable fluorescent dye. Two copolymers differing in polymer chain structure (linear and star-like) and molecular weight (30 and 200kDa) were used to distinguish between carriers with molecular weights above and below the limit for renal filtration. The rate of model drug release from the conjugates was determined in vitro. The biodistributions of the six most promising conjugates were investigated in vivo in athymic nude mice inoculated with human colon carcinoma xenograft. The structure of the spacer in the vicinity of the hydrazone bond significantly influenced the release rate of the model drug. The slow release rate of a pyridyl group bearing spacer resulted in a greater amount of the model drug being transported to the tumor, which was independent of the carrier structure. The results of this study emphasize the importance of careful selection of the structure and appropriate spacer when designing polymer conjugates intended for passive tumor targeting.

Oncolytic adenoviruses (Ads) selectively replicate within and destroy cancer cells, making them one of the most powerful therapeutics available. However, all robust responses reported to date (1–3) have relied on intratumoral injection, and until Ads can achieve efficacy following intravenous delivery their clinical use will be restricted. In particular, systemic delivery of Ads to treat metastatic cancer will require improved circulation kinetics, extravasation from the bloodstream into the tumor, and intratumoral penetration. After intravenous delivery, Ads bind antibodies, complement, and blood cells (4,5). In response, “stealthing” technologies have been developed, whereby Ads are coated with biocompatible polymers such as polyethylene glycol or N-(2-hydroxypropyl)methacrylamide (6–9). Stealthing removes natural Ad tropism and prevents binding to blood components, leading to extended circulation (10). However, by stealthing Ads to provide sufficient protection during delivery, their ability to infect cells upon arrival in tumors is often compromised. To meet this challenge, polymers can be designed to selectively degrade upon exposure to the tumor milieu. This is possible because disregulated growth of cells within tumors creates a hypoxic and low pH environment that is distinct from normal tissue (11,12). We report the development of a coating polymer that enhances circulation and also allows triggered uncoating and reactivation of Ads within tumors. Despite benefiting from the enhanced permeability retention effect (13), the passage of stealthed Ads from the bloodstream into tumors is still suboptimal, with less than 0.1% of dose achieving deposition in the tumor (4). Furthermore, the intratumoral distribution of Ads remains exclusively perivascular, and without penetration deep into the tumor, the therapeutic effect is restricted to a small proportion of its mass. Such limitations are shared by other therapeutics, such as drug–polymer conjugates and liposomes (14–17). In response, external stimuli have been applied to improve uptake into and spread through tumors (18–21). We feel that focused ultrasound is the safest and most clinically applicable of these strategies. Indeed, our in vitro studies have shown that ultrasound can provide a powerful stimulus to propel Ads deep into tumor-mimicking material (22). To achieve these effects, Ads were coadministered with a microbubble formulation (SonoVue [SV]), which provided nuclei for the initiation of a phenomenon known as inertial cavitation. This term describes the expansion, contraction, and violent collapse of a bubble in response to ultrasound, which creates microstreaming and shock waves. Such events can be used to move macromolecules more than 200 µm (21). The use of microbubbles and ultrasound to improve Ad delivery to tumors is ideal because the ultrasound pressures required are modest and clinically applicable and the inertial cavitation events created are targetable and produce distinct nonharmonic emissions that can be mapped, providing valuable feedback on the success of the procedure (23). We have improved the efficacy of Ads by combining stealthing and ultrasound to overcome their poor pharmacokinetics and limited extravasation and penetration into the tumor.

The synthesis, characterization and results of evaluation of the biological behavior of HPMA copolymer conjugates bearing anti-cancer drugs doxorubicin and mitomycin C are described. Two HPMA copolymer carrier types were synthesized: the linear copolymer and the biodegradable high-molecular-weight diblock copolymer containing a degradable disulfide bond. The polymer-drug conjugates incubated in buffers modeling the intracellular environment released the drugs more rapidly than those incubated in bloodstream conditions. Significant in vitro and in vivo antitumor synergistic activity of the conjugates in the treatment of EL-4 T-cell demonstrates their high potential for solid tumor treatment.

Polymer conjugates of anticancer drugs have shown high potential for assisting in cancer treatments. The pH-labile spacers allow site-specific triggered release of the drugs. We synthesized and characterized model drug conjugates with hydrazide bond-containing poly[N-(2-hydroxypropyl)methacrylamide] differing in the chemical surrounding of the hydrazone bond-containing spacer to find structure–drug release rate relationships. The conjugate selected for further studies shows negligible drug release in a pH 7.4 buffer but released 50% of the ellipticinium drug within 24 h in a pH 5.0 phosphate saline buffer. The ellipticinium drug retained the antiproliferative activity of the ellipticine.

The high affinity and specificity between avidin and biotin were employed to bind a recombinant single-chain antibody fragment to synthetic hydrophilic polymer drug carriers. Two semitelechelic polymers, based on poly(ethylene glycol) and poly[ N-(2-hydroxypropyl)methacrylamide], each containing a single thiol end group, were conjugated to dithiopyridyl-modified avidin. The biotinylated recombinant single-chain antibody fragment of the M75 antibody was then noncovalently bound to the polymer-avidin conjugates. The recombinant protein was chosen as a targeting ligand against carbonic anhydrase IX, a marker overexpressed by tumor cells of various human carcinomas. The antigen-binding affinity of the polymer–single-chain antibody fragment complex was confirmed by enzyme-linked immuno sorbent assay (ELISA). This approach provides an original, nondestructive way of preparing supramolecular systems intended for targeted delivery of therapeutics utilizing modern chemical procedures, including reversible addition–fragmentation chain-transfer polymerization and recombinant DNA techniques.

Fine needle aspiration biopsy (FNAB) is an easy method with an option of repetitive withdrawal of cell material.First, mice were inoculated with mouse T-lymphoma, after 10 d the samples from tumor, lymph nodes and spleen gained by FNAB and excision were analyzed by flow cytometry. Tumor progression was compared to the control group simultaneously. Then, 10 d after tumor cell inoculation free doxorubicin (DOX) or different PHPMA DOX conjugates were injected. Cell material was analyzed to detect subpopulations of lymphocyte infiltrate, and levels of cytokines in correlation with progression or regression of the disease.FNAB has no influence on the tumor's growth or survival of experimental animals. After treatment with PHPMA conjugates there was a significant increase of T-lymphocyte subpopulations in tumor microenvironment compared to controls or free DOX, but only in mice with confirmed macroscopic regression of tumor within two weeks. Mice treated with conjugates showed significantly lower cancer infiltration of lymph nodes and spleen.FNAB provides a great benefit to in vivo monitoring of cell changes directly in the tumor after treatment. The number of infiltrating T-lymphocytes increases in correlation with consecutive tumor eradication after treatment with PHPMA. This proves that not only direct cytotoxic but also imunostimulating effect are necessary for successful treatment.

We synthesized N-(2-hydroxypropyl)methacrylamide polymer conjugated with zinc protoporphyrin (HPMA-ZnPP) and evaluated its application for tumor detection by imaging and treatment by light exposure using in mouse sarcoma model. To characterize HPMA-ZnPP micelle, we measured its micellar size, surface charge, stability, photochemical, biochemical properties and tissue distribution. In vivo anti-tumor effect and fluorescence imaging were carried out to validate the tumor selective accumulation and therapeutic effect by inducing singlet oxygen by light exposure. HPMA-ZnPP was highly water soluble and formed micelles spontaneously having hydrophobic clustered head group of ZnPP, in aqueous solution, with a hydrodynamic diameter of 82.8±41.8 nm and zeta-potential of +1.12 mV. HPMA-ZnPP had a long plasma half-life and effectively and selectively accumulated in tumors. Although HPMA-ZnPP alone had no toxicity in S-180 tumor-bearing mice, light-irradiation significantly suppressed tumor growth in vivo, similar to the cytotoxicity to HeLa cells in vitro upon endoscopic light-irradiation. HPMA-ZnPP can visualize tumors by fluorescence after i.v. injection, which suggests that this micelle may be useful for both tumor imaging and therapy. Here we describe preparation of a new fluorescence nanoprobe that is useful for simultaneous tumor imaging and treatment, and application to fluorescence endoscopy is now at visible distance.

Various different passively and actively targeted nanomedicines have been designed and evaluated over the years, in particular for the treatment of cancer. Reasoning that the potential of ligand-modified nanomedicines can be substantially improved if intrinsically active targeting moieties are used, we have here set out to assess the in vivo efficacy of nanobody-modified core-crosslinked polymeric micelles containing covalently entrapped doxorubicin. Nanobody-modified polymeric micelles were found to inhibit tumor growth even in the absence of a drug, and nanobody-modified micelles containing doxorubicin were significantly more effective than nanobody-free micelles containing doxorubicin. Based on these findings, we propose that the combination of two therapeutic strategies within one nanomedicine formulation, i.e. the intrinsic pharmacological activity of ligand-modified carrier materials with the cytostatic activity of the incorporated chemotherapeutic agents, is a highly promising approach for improving the efficacy of tumor-targeted combination therapy.

Background Site-specific gene delivery requires vectors that combine stability in the delivery phase with substantial biological activity within target cells. The use of biological trigger mechanisms provides one promising means to achieve this, and here we report a transfection trigger mechanism based on intracellular reduction. Methods Plasmid DNA was condensed with thiolated polyethylenimine (PEI-SH) and the resulting nanoparticles surface-coated using thiol-reactive poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) with 2-pyridyldisulfanyl or maleimide groups, forming reducible disulphide-linked or stable thioether-linked coatings, respectively. Results Both sets of polymer-coated complexes had similar size and were stable to a 250-fold excess of the polyanion poly(aspartic acid) (PAA). Reduction with dithiothreitol (DTT) allowed complete release of DNA from disulphide-linked coated complexes, whereas complexes with thioether-linked coating remained stable. Disulphide-linked complexes showed 40–100-fold higher transfection activity than thioether-linked ones, and activity was selectively further enhanced by boosting intracellular glutathione using glutathione monoethyl ester or decreased using buthionine sulfoximine. The chloroquine- and serum-independent transfection activity of disulphide-linked coated complexes suggests this system may provide a viable trigger mechanism to enable site-specific transfection in complex biological settings. Conclusions Linkage of hydrophilic polymer coating to PEI/DNA complexes via reducible disulphide bonds offers a means of fulfilling the contradictory requirements for extracellular stability and intracellular activity. Copyright © 2004 John Wiley & Sons, Ltd.

Adenovirus provides many opportunities as a vector for delivery of cytotoxic genes to tumours. Polymer coating of adenovirus is known to increase its plasma circulation kinetics, affording the possibility of active and passive targeting to tumours. Here we show that polymer-coating adenovirus (pc-virus) abrogates its normal infectivity in vitro and also in liver following intravenous injection. The coated virus accumulates within solid subcutaneous AB22 mesothelioma tumours 40-times more than unmodified virus, and mediates higher levels of transgene expression within tumours. This is the first demonstration of passive tumour targeting of polymer-coated adenoviruses administered by intravenous injection, and also the first time pc-virus has been shown to be infectious following passive targeting to tumours in vivo. This technology provides an interesting option for delivery of therapeutic viruses to disseminated tumour masses by intravenous injection.

Nanomedicines have provided fresh impetus in the fight against cancer due to their selectivity and power. However, these agents are limited when delivered intravenously due to their rapid clearance from the bloodstream and poor passage from the bloodstream into target tumours. Here we describe a novel stealthing strategy which addresses both these limitations and thereby demonstrate that both the passive and mechanically-mediated tumour accumulation of the model nanomedicine adenovirus (Ad) can be substantially enhanced. In our strategy gold nanoparticles were thoroughly modified with 2 kDa polyethyleneglycol (PEG) and then linked to Ad via a single reduction-cleavable 5 kDa PEG. The resulting Ad–gold–PEG construct was compared to non-modified Ad or conventionally stealthed Ad–poly[N-(2-hydroxypropyl)methacrylamide] (Ad–PHPMA). Notably, although Ad–gold–PEG was of similar size and surface charge to Ad–PHPMA the increase in density, resulting from the inclusion of the gold nanoparticles, provided a substantial enhancement of ultrasound-mediated transport. In an in vitro tumour mimicking phantom, the level and distance of Ad–gold–PEG transport was shown to be substantially greater than achieved with Ad–PHPMA. In in vivo studies 0.1% of an unmodified Ad dose was shown to accumulate in tumours, whereas over 12% of the injected dose was recovered from the tumours of mice treated with Ad–gold–PEG and ultrasound. Ultimately, a significant increase in anti-tumour efficacy resulted from this strategy. This stealthing and density-increasing technology could ultimately enhance clinical utility of intravenously delivered nanoscale medicines including viruses, liposomes and antibodies.

The enhanced permeability and retention (EPR) effect, a tumor-targeting principle of nanomedicine, serves as a standard for tumor-targeted anticancer drug design. There are 3 key issues in ideal EPR-based antitumor drug design: i) stability in blood circulation; ii) tumor-selective accumulation (EPR effect) and efficient release of the active anticancer moiety in tumor tissues; and iii) the active uptake of the active drug into tumor cells. Using these principles, we developed N-(2- hydroxypropyl)methacrylamide (HPMA) copolymer-conjugated pirarubicin (P-THP), which uses hydrazone bond linkage; it was shown to exhibit prolonged circulation time, thereby resulting in good tumor-selective accumulation. More importantly, the hydrazone bond ensured selective and rapid release of the active drug, pirarubicin (THP), in acidic tumor environments. Further, compared to other anthracycline anticancer drugs (eg, doxorubicin), THP demonstrated more rapid intracellular uptake. Consequently, P-THP showed remarkable antitumor effect with minimal side effects. In a clinical pilot study of a stage IV prostate cancer patient with multiple metastases in the lung and bone, P-THP (50-75 mg administered once every 2-3 weeks) was shown to clear the metastatic nodules in the lung almost completely after 3 treatments where 50-70 mg THP equivalent each was administerd per 70 kg body wt, and bone metastasis disappeared after 6 months. There was no recurrence after 2 years. The patient also retained an excellent quality of life during the treatment without any apparent side effects. Thus, we propose the clinical development of P-THP as an EPR-based tumor-targeted anticancer drug.

The molecular weight and molecular architecture of soluble polymer drug carriers significantly influence the biodistribution and anti-tumour activities of their doxorubicin (DOX) conjugates in tumour-bearing mice. Biodistribution of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-DOX conjugates of linear and star architectures were compared in EL4 T-cell lymphoma-bearing mice. Biodistribution, including tumour accumulation, and anti-tumour activity of the conjugates strongly depended on conjugate molecular weight (MW), polydispersity, hydrodynamic radius (R(h)) and molecular architecture. With increasing MW, renal clearance decreased, and the conjugates displayed extended blood circulation and enhanced tumour accumulation. The linear conjugates with flexible polymer chains were eliminated by kidney clearance more quickly than the highly branched star conjugates with comparable MWs. Interestingly, the data suggested different mechanisms of renal filtration for star and linear conjugates. Only star conjugates with MWs below 50,000g.mo(-1) were removed by kidney filtration, while linear polymer conjugates with MWs near 70,000g.mol(-1), exceeding the generally accepted limit for renal elimination, were detected in the urine 36-96h after injection. Additionally, survival of tumour-bearing mice was strongly dependent on molecular weight and polymer conjugate architecture. Treatment of mice with the lower MW conjugate at a dose of 10mg DOX eq./kg resulted in 12% long-term surviving animals, while treatment with the corresponding star conjugate enabled 75% survival of animals.

Intravenous delivery of therapeutic virus particles remains a major goal for virotherapy of metastatic cancer. Avoiding phagocytic capture and unwanted infection of nontarget cells is essential for extended plasma particle kinetics, and simply ablating one or the other does not give extended plasma circulation. Here we show that polymer coating of adenovirus type 5 (Ad5) can combine with predosing strategies or Kupffer cell ablation to achieve systemic kinetics with a half-life >60 min, allowing ready access to peripheral tumors. Accumulation of virus particles within tumor nodules is proportional to the area under the plasma concentration/time curve. Polymer coating wild-type Ad5 in this way is known to decrease hepatic toxicity, increasing the dose of virus particles that can be safely administered. Using polymer-coating technology to deliver a replicating Ad5 systemically, virus replication and transgene expression was almost totally confined to tumor tissues, giving a much improved therapeutic index compared with uncoated virus, and complete control of human HepG2 tumor xenografts.

Nanomedicines are sub-micrometer-sized carrier materials designed to improve the biodistribution of i.v. administered (chemo-) therapeutic agents. In recent years, ever more efforts in the nanomedicine field have employed optical imaging (OI) techniques to monitor biodistribution and target site accumulation. Thus far, however, the longitudinal assessment of nanomedicine biodistribution using OI has been impossible, due to limited light penetration (in the case of 2D fluorescence reflectance imaging; FRI) and to the inability to accurately allocate fluorescent signals to nonsuperficial organs (in the case of 3D fluorescence molecular tomography; FMT). Using a combination of high-resolution microcomputed tomography (μCT) and FMT, we have here set out to establish a hybrid imaging protocol for noninvasively visualizing and quantifying the accumulation of near-infrared fluorophore-labeled nanomedicines in tissues other than superficial tumors. To this end, HPMA-based polymeric drug carriers were labeled with Dy750, their biodistribution and tumor accumulation were analyzed using FMT, and the resulting data sets were fused with anatomical μCT data sets in which several different physiologically relevant organs were presegmented. The robustness of 3D organ segmentation was validated, and the results obtained using 3D CT-FMT were compared to those obtained upon standard 3D FMT and 2D FRI. Our findings convincingly demonstrate that combining anatomical μCT with molecular FMT facilitates the noninvasive assessment of nanomedicine biodistribution.

We report a rigorous investigation into the detailed structure of nanoparticles already shown to be successful drug delivery nanocarriers. The basic structure of the drug conjugates consists of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond and a defined amount of cholesterol moieties that vary in hydrophobicity. The results show that size, anisotropy, and aggregation number N(aggr) of the nanoparticles grows with increasing cholesterol content. From ab initio calculations, we conclude that the most probable structure of HPMA copolymer-cholesterol nanoparticles is a pearl necklace structure, where ellipsoidal pearls mainly composed of cholesterol are covered by a HPMA shell; pearls are connected by bridges composed of hydrophilic HPMA copolymer chains. Using a combination of techniques, we unambiguously show that the Dox moieties are not impregnated inside a cholesterol core but are instead uniformly distributed across the whole nanoparticle, including the hydrophilic HPMA shell surface.

Acridines are potent DNA-intercalating anticancer agents with high in vivo anticancer effectiveness, but also severe side effects. We synthesized five 9-anilinoacridine-type drugs and their conjugates with biocompatible water-soluble hydrazide polymer carrier. All of the synthesized acridine drugs retained their in vitro antiproliferative properties. Their polymer conjugates were sufficiently stable at pH 7.4 (model of pH in blood plasma) while releasing free drugs at pH 5.0 (model of pH in endosomes). After internalization of the conjugates, the free drugs were released and are visible in cell nuclei by fluorescence microscopy. Their intercalation ability was proven using a competitive ethidium bromide displacement assay.

Various biodegradable micellar polymer drug carriers based on N-(2-hydroxypropyl)methacrylamide copolymers and their conjugates with doxorubicin are synthesized. The conjugates are designed to treat solid tumors and subsequently to allow carrier elimination from the body. All copolymers self-assemble in aqueous solution into supramolecular structures with highly hydrophobic cholesterol derivatives in the core and doxorubicin located in the hydrophilic shell. The drug is released quickly from the micelles incubated at pH = 5.0 (modelling the endosomes of tumor cells). Slower release of cholesterol derivatives leading to disintegration of the micelles is observed.

Potentially biodegradable graft polymer core-shell nanoparticles assembling by addition of metal ions intended to be used for radionuclide and drug delivery purposes are described. With Cu2+ ions, the self-assembly is slow (within minutes) with low efficiency. With Fe3+ ions the nanoparticles are formed immediately and are of convenient size for passive tumor accumulation by the enhanced permeation and retention effect. Full in vitro degradation of these particles is achieved with deferoxamine as a model of in vivo transchelation capacity.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer—drug conjugates bearing the antiinflammatory and anti-proliferative drug dexamethasone (DEX) and the anti-cancer drug doxorubicin (DOX) bound to either the polymer carrier separately or in combination, were synthesized. Both the in vitro and in vivo anti-cancer activity on mouse B-cell (38C13) and T-cell (EL4) lymphoma was studied. The conjugates were fairly stable in model buffers at pH 7.4, simulating blood pH, and the drug was released by chemical hydrolysis at pH values based on the endosomal and lysosomal environments in the target cells (pH 5—6). As a control, polymer conjugates containing only a single-drug (DOX or DEX) attached to the HPMA copolymer carrier were synthesized and tested. Contrary to the treatment with either single drug conjugate, where no significant long-term survival was observed, a synergistic effect of the two drugs was manifested in vivo. Treatment of mice bearing B-cell lymphoma 38C13 formulated as a mixture of both single drug-containing conjugates or a as a conjugate with both drugs in combination gave long-term survivors.

Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. Tumour-associated endothelium offers an additional mechanism for enhanced viral uptake into tumours which is accessible for systemic gene delivery. Building on expertise in using polymer ‘stealthed’ viruses for targeting in vivo, adenovirus expressing luciferase (Adluc) was coated with an amino-reactive polymer based on poly [N-(2-hydroxypropyl) methacrylamide] to ablate normal infection pathways. Direct linkage of a monoclonal antibody against E-selectin (MHES) demonstrated E-selectin-specific transduction of tumour necrosis factor-α (TNF-α)-activated endothelial cells. A two-component targeting system using protein G was developed, to provide optimal antibody orientation. We report an enhancement in transduction of TNF-α-activated endothelium in vitro and ex vivo in a human umbilical vein cord model using the MHES antibody. Similarly a virus retargeted using a chimeric P-selectin Glycoprotein Ligand-1-Fc fusion (PSGL-1) protein showed better circulation kinetics and significant uptake into HepG2 xenografts following systemic administration in mice, with 36-fold higher genome copies, compared with non-modified virus. Immunohistochemistry staining of tumour sections from mice treated with PSGL-1-retargeted virus showed a co-localisation of firefly luciferase with CD31 suggesting selective endothelial targeting. Employment of optimal viral modification using protein G will enable exploration and comparison of alternative targeting ligands targeting tumour-associated endothelium., Graphical Abstract

To assist in overcoming the inherent instability of nucleic acid-containing polyplexes in physiological solutions, we have here set out to develop removable nanocoatings for modifying the surface of siRNA-based nanoparticles. Here, N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymers containing carbonylthiazolidine-2-thione (TT) reactive groups in their side chains bound via disulfide spacers to the polymeric backbone were synthesized, and these copolymers were used to coat the surface of polyplexes formed by the self-assembly of anti-Luciferase siRNA with the polycations polyethylene imine (PEI) and poly(HPMA)-grafted poly(l-lysine) (GPL). The coating process was monitored by analyzing changes in the weight-average molecular weight (M(w)), the hydrodynamic radius (R(h)), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to the surface of the polyplexes, forming a ∼5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing anti-Luciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.

To assist intravenously administered anticancer agents in achieving proper circulation times and tumor concentrations, and to thereby improve the balance between their efficacy and their toxicity, a large number of drug delivery systems have been designed and evaluated over the years. Clinically relevant examples of such nanometer-sized carrier materials are liposomes, polymers, micelles and antibodies. In the vast majority of cases, however, and especially in patients, nanomedicine formulations are only able to attenuate the toxicity of the conjugated or entrapped chemotherapeutic drug, and they generally fail to improve the efficacy of the intervention. To overcome this shortcoming, and to broaden the clinical applicability of tumor-targeted nanomedicines, in the past 5 years we have developed several concepts for using N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer therapeutics to enhance the efficacy of combined modality anticancer therapy. Regarding surgery, HPMA copolymers were shown to be able to improve the retention of intratumorally administered chemotherapeutic agents at the pathological site, and to thereby increase their therapeutic index. Regarding radiotherapy, a synergistic interaction was observed, with radiotherapy improving the tumor accumulation of the copolymers, and with copolymers improving both the efficacy and the tolerability of radiochemotherapy. Futhermore, regarding chemotherapy combinations, we have for the first time provided in vivo evidence showing that passively tumor-targeted polymeric drug carriers can be used to deliver two different drugs to tumors simultaneously. Based on these findings, and on the fact that the concepts developed are considered to be broadly applicable, we conclude that nanomedicine formulations are highly suitable systems for improving the efficacy of combined modality anticancer therapy.