Augmentation of EPR Effect and Efficacy of Anticancer Nanomedicine by Carbon Monoxide Generating Agents.

One obstacle to the successful delivery of nanodrugs into solid tumors is the heterogeneity of an enhanced permeability and retention (EPR) effect as a result of occluded or embolized tumor blood vessels. Therefore, the augmentation of the EPR effect is critical for satisfactory anticancer nanomedicine. In this study, we focused on one vascular mediator involved in the EPR effect, carbon monoxide (CO), and utilized two CO generating agents, one is an extrinsic CO donor (SMA/CORM2 micelle) and another is an inducer of endogenous CO generation via heme oxygenase-1 (HO-1) induction that is carried out using pegylated hemin. Both agents generated CO selectively in solid tumors, which resulted in an enhanced EPR effect and a two- to three-folds increased tumor accumulation of nanodrugs. An increase in drug accumulation in the normal tissue did not occur with the treatment of CO generators. In vivo imaging also clearly indicated a more intensified fluorescence of macromolecular nanoprobe in solid tumors when combined with these CO generators. Consequently, the combination of CO generators with anticancer nanodrugs resulted in an increased anticancer effect in the different transplanted solid tumor models. These findings strongly warrant the potential application of these CO generators as EPR enhancers in order to enhance tumor detection and therapy using nanodrugs.

Impact of Polymer-TLR-7/8 Agonist (Adjuvant) Morphology on the Potency and Mechanism of CD8 T Cell Induction.

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.

Augmentation of the Enhanced Permeability and Retention Effect with Nitric Oxide-Generating Agents Improves the Therapeutic Effects of Nanomedicines.

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.

N-(2-hydroxypropyl)methacrylamide polymer conjugated pyropheophorbide-a, a promising tumor-targeted theranostic probe for photodynamic therapy and imaging.

Tumor-targeted photodynamic therapy (PDT) using polymeric photosensitizers is a promising therapeutic strategy for cancer treatment. In this study, we synthesized a pHPMA conjugated pyropheophorbide-a (P-PyF) as a cancer theranostic agent for PDT and photodynamic diagnostics (PDD). Pyropheophorbide-a has one carboxyl group which was conjugated to pHPMA via amide bond yielding the intended product with high purity. In aqueous solutions, P-PyF showed a mean particle size of ∼200 nm as it forms micelle which exhibited fluorescence quenching and thus very little singlet oxygen (1O2) production. In contrast, upon disruption of micelle strong fluorescence and 1O2 production were observed. In vitro study clearly showed the PDT effect of P-PyF. More potent 1O2 production and PDT effect were observed during irradiation at ∼420 nm, the maximal absorbance of pyropheophorbide-a, than irradiation at longer wavelength (i.e., ∼680 nm), suggesting selection of proper absorption light is essential for successful PDT. In vivo study showed high tumor accumulation of P-PyF compared with most of normal tissues due to the enhanced permeability and retention (EPR) effect, which resulting in superior antitumor effect under irradiation using normal xenon light source of endoscope, and clear tumor imaging profiles even in the metastatic lung cancer at 28 days after administration of P-PyF. On the contrary irradiation using long wavelength (i.e., ∼680 nm), the lowest Q-Band, exhibited remarkable tumor imaging effect with little autofluorescence of background. These findings strongly suggested P-PyF may be a potential candidate-drug for PDT/PDD, particularly using two different wavelength for treatment and detection/imaging, respectively.

Bloodstream Stability Predetermines the Antitumor Efficacy of Micellar Polymer-Doxorubicin Drug Conjugates with pH-Triggered Drug Release.

Herein, the biodegradable micelle-forming amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA)-based polymer conjugates with the anticancer drug doxorubicin (Dox) designed for enhanced tumor accumulation were investigated, and the influence of their stability in the bloodstream on biodistribution, namely, tumor uptake, and in vivo antitumor efficacy were evaluated in detail. Dox was attached to the polymer carrier by a hydrazone bond enabling pH-controlled drug release. While the polymer-drug conjugates were stable in a buffer at pH 7.4 (mimicking bloodstream environment), Dox was released in a buffer under mild acidic conditions modeling the tumor microenvironment or cells. The amphiphilic polymer carriers differed in the structure of hydrophobic cholesterol derivative moieties bound to the HPMA copolymers via a hydrolyzable hydrazone bond, exhibiting different rates of micellar structure disintegration at various pH values. Considerable dependence of the studied polymer-drug conjugate biodistribution on the stability of the micellar structure was observed in neutral, bloodstream-mimicking, environment, showing that a faster rate of the micelle disintegration in pH 7.4 increased the conjugate blood clearance, decreased tumor accumulation, and significantly reduced the tumor:blood and tumor:muscle ratios. Similarly, the final therapeutic outcome was strongly affected by the stability of the micellar structure because the most stable micellar conjugate showed an almost similar therapeutic outcome as the water-soluble, nondegradable, high-molecular-weight starlike HPMA copolymer-Dox conjugate, which was highly efficient in the treatment of solid tumors in mice. Based on the results, we conclude that the bloodstream stability of micellar polymer-anticancer drug conjugates, in addition to their low side toxicity, is a crucial parameter for their efficient solid tumor accumulation and high in vivo antitumor activity.

HPMA copolymer conjugate with pirarubicin: In vitro and ex vivo stability and drug release study.

We have developed a tumor environment-responsive polymeric anticancer prodrug containing pirarubicin (THP) conjugated to N-(2-hydroxypropyl) methacrylamide copolymer (PHPMA), [P-THP], through a spacer containing pH-sensitive hydrazone bond, that showed remarkable therapeutic effect against various tumor models and in a human pilot study. Toward clinical development, here we report THP release profile from its HPMA copolymer conjugate, the conjugate stability, protein and cell-binding and solubility of P-THP. Size exclusion chromatography of P-THP (molecular weight 38 kDa) showed similar hydrodynamic volume as bovine serum albumin (BSA) in aqueous solution, with no apparent interactions with BSA, nor aggregation by itself. pH-responsive release of free THP was reconfirmed at pHs 6.5 and lower. The drug release was significantly affected by a type of used buffer. Phosphate buffer seems to facilitate faster hydrazone bond cleavage at pH 7.4 whereas higher stability was achieved in L-arginine solution which yielded only little cleavage and THP release, approx. 15% within 2 weeks at the same pH at 25 °C. Furthermore, ex vivo study using sera of different animal species showed very high stability of P-THP. Incubation with blood showed high stability of P-THP during circulation, without binding to blood cells. These findings revealed that L-arginine solution provides appropriate media for formulation of P-THP infusion solution as tumor-targeted polymeric anticancer drug based on EPR effect.

HPMA Copolymer-Drug Conjugates with Controlled Tumor-Specific Drug Release.

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.

Comparison of the pharmacological and biological properties of HPMA copolymer-pirarubicin conjugates: A single-chain copolymer conjugate and its biodegradable tandem-diblock copolymer conjugate.

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 Mw below the renal threshold (39g/mol) (named LINEAR) and the disulfide-linked tandem-polymeric dimer of the poly(HPMA)-pirarubicin conjugate with an Mw above the renal threshold (93g/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 24h) 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 150days), without any apparent toxicity in an S180 tumor model. However, the LD50 value of LINEAR was slightly higher than that of DIBLOCK (50mg/kg vs 37.5mg/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.

Pronounced Cellular Uptake of Pirarubicin versus That of Other Anthracyclines: Comparison of HPMA Copolymer Conjugates of Pirarubicin and Doxorubicin.

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.

Biodegradable Micellar HPMA-Based Polymer-Drug Conjugates with Betulinic Acid for Passive Tumor Targeting.

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.

[Missile-Type Tumor-Targeting Polymer Drug, P-THP, Seeks Tumors via Three Different Steps Based on the EPR Effect].

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.

Overcoming multidrug resistance in Dox-resistant neuroblastoma cell lines via treatment with HPMA copolymer conjugates containing anthracyclines and P-gp inhibitors.

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 16h 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 Drug Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent Approaches, Release Control, and Clinical Studies.

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.

Fluorescent boronate-based polymer nanoparticles with reactive oxygen species (ROS)-triggered cargo release for drug-delivery applications.

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.

iBodies: Modular Synthetic Antibody Mimetics Based on Hydrophilic Polymers Decorated with Functional Moieties.

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.

HPMA Copolymer-Conjugated Pirarubicin in Multimodal Treatment of a Patient with Stage IV Prostate Cancer and Extensive Lung and Bone Metastases.

Nanomedicine allows achievement of tumor-selective drug delivery because of the enhanced permeability and retention (EPR) effect of solid tumors. We report here the first clinical application of a new agent-HPMA copolymer-conjugated pirarubicin (P-THP)-with a molecular size of about 8 nm, or 38.5 kDa. A patient had advanced prostate cancer with multiple metastases in the lung, pelvis, femur, and perhaps the sacrum. In April 2013, this 60-year-old patient started treatment with leuprorelin and estradiol, which continued until July 2014, but the patient became refractory to this treatment. So the patient underwent proton beam radiotherapy targeted to the primary prostate cancer, and P-THP was administered for numerous metastatic tumor nodules concomitantly with radiotherapy. This combination therapy had remarkable results, with complete remission of multiple metastases in the lung and bone. The prostate-specific antigen (PSA) value was decreased from about 1000 ng/mL on April 30, 2013, to about 100 ng/mL on June 24, 2013, with hormone therapy, but rose again to 964.2 ng/mL and then to 1472 ng/mL in July 2013, during leuprorelin administration. P-THP treatment administered concomitantly with proton beam irradiation was started in August 2013. The PSA value was decreased to 102 ng/mL on August 26, 2013, and then to 0.971 ng/mL on October 8, 2013, and 0.277 ng/mL on January 15, 2015. The P-THP doses ranged from 30 to 75 mg of free THP equivalent/patient every 2-3 weeks without signs of serious toxicity, such as cardiovascular side effects or a reduction in quality of life. No evidence of relapse was found more than 20 months after P-THP administration. This case demonstrates the value of hydrazone-bonded polymeric drugs in multimodal therapy.

The structure-dependent toxicity, pharmacokinetics and anti-tumour activity of HPMA copolymer conjugates in the treatment of solid tumours and leukaemia.

Polymer drug carriers that are based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been widely used in the development and synthesis of high-molecular-weight (HMW) drug delivery systems for cancer therapy. In this study, we compared linear (Mw ~27kDa, Rh ~4nm) and non-degradable star (Mw ~250kDa, Rh ~13nm) HPMA copolymer conjugates bearing anthracycline antibiotic doxorubicin (DOX) bound via pH-sensitive hydrazone bond. We determined the in vitro and in vivo toxicity of both conjugates and their maximum tolerated dose (MTD). We also compared their anti-tumour activity in mouse B-cell leukaemia (BCL1) and a mouse T-cell lymphoma (EL4) model. We found that MTD was higher for the linear conjugate (85mgDOX/kg) and lower for the star conjugate (22.5mgDOX/kg). An evaluation of the intestinal barrier integrity using FITC-dextran as a gut permeability tracer proved that no pathology was caused by the MTD of either conjugate. However, free DOX showed some damage to the gut barrier. The therapy of BCL1 leukaemia by both of the polymeric conjugates using the MTD or its fraction (i.e., equitoxic dosage) showed better results in the case of the star conjugate. On the other hand, treatment of EL4 lymphoma seemed to be more efficient when the linear conjugate was used. We suppose that the anti-cancer treatment of solid tumours and leukaemias requires different types of drug conjugates. We hypothesise that the most suitable HPMA copolymer-DOX conjugate for the treatment of solid tumours should have an HMW structure with increased Rh that would be stable for three to four days after the conjugate administration and then rapidly disintegrate in the short polymer chains, which are excretable from the body by glomerular filtration. On the other hand, the treatment of leukaemia requires a drug conjugate with a long circulation half-life. This would provide an active drug, whilst slowly degrading to excretable fragments.

Novel IL-2-Poly(HPMA)Nanoconjugate Based Immunotherapy.

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.

Thermoresponsive Polymer Micelles as Potential Nanosized Cancerostatics.

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.

Increasing the density of nanomedicines improves their ultrasound-mediated delivery to tumours.

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 2kDa polyethyleneglycol (PEG) and then linked to Ad via a single reduction-cleavable 5kDa 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.