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1.
Biomacromolecules ; 20(2): 854-870, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30608149

RESUMO

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.


Assuntos
Adjuvantes Imunológicos/química , Linfócitos T CD8-Positivos/efeitos dos fármacos , Ativação Linfocitária , Micelas , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Feminino , Hidrodinâmica , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica
2.
Mol Pharm ; 15(9): 3654-3663, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-29543465

RESUMO

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.


Assuntos
Doxorrubicina/química , Doxorrubicina/farmacocinética , Polímeros/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Feminino , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Linfoma/sangue , Linfoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Micelas
3.
Chem Rev ; 116(9): 5338-431, 2016 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-27109701

RESUMO

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.


Assuntos
Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Nanopartículas de Magnetita/química , Polímeros/química , Portadores de Fármacos/química , Concentração de Íons de Hidrogênio , Ligantes , Porosidade , Propriedades de Superfície
4.
Mol Pharm ; 13(12): 4106-4115, 2016 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-27934482

RESUMO

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.


Assuntos
Acrilamidas/química , Antibióticos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Polímeros/química , Animais , Antibióticos Antineoplásicos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Doxorrubicina/química , Portadores de Fármacos/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Polímeros/administração & dosagem , Sarcoma Experimental/tratamento farmacológico , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Biomacromolecules ; 17(11): 3493-3507, 2016 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-27636143

RESUMO

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.


Assuntos
Proliferação de Células/efeitos dos fármacos , Doxorrubicina/química , Metacrilatos/química , Neoplasias/tratamento farmacológico , Animais , Plásticos Biodegradáveis/química , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Humanos , Metacrilatos/administração & dosagem , Camundongos , Micelas , Triterpenos Pentacíclicos , Polímeros/administração & dosagem , Polímeros/química , Triterpenos/administração & dosagem , Triterpenos/química , Ácido Betulínico
6.
Gan To Kagaku Ryoho ; 43(5): 549-57, 2016 May.
Artigo em Japonês | MEDLINE | ID: mdl-27210083

RESUMO

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.


Assuntos
Antineoplásicos/uso terapêutico , Permeabilidade da Membrana Celular , Neoplasias/tratamento farmacológico , Polímeros/uso terapêutico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Ensaios Clínicos como Assunto , Humanos , Polímeros/efeitos adversos , Polímeros/química , Microambiente Tumoral
7.
Angew Chem Int Ed Engl ; 55(7): 2356-60, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26749427

RESUMO

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.


Assuntos
Anticorpos/química , Mimetismo Molecular , Polímeros/química , Linhagem Celular Tumoral , Humanos , Interações Hidrofóbicas e Hidrofílicas
8.
Biomacromolecules ; 16(8): 2493-505, 2015 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-26153904

RESUMO

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.


Assuntos
Antineoplásicos/administração & dosagem , Doxorrubicina/análogos & derivados , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Antineoplásicos/química , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Micelas , Nanopartículas/química , Polímeros/administração & dosagem , Polímeros/química
9.
Nano Lett ; 14(2): 972-81, 2014 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-24422585

RESUMO

Enhanced permeability and retention (EPR) and the (over-) expression of angiogenesis-related surface receptors are key features of tumor blood vessels. As a consequence, EPR-mediated passive and Arg-Gly-Asp (RGD) and Asn-Gly-Arg (NGR) based active tumor targeting have received considerable attention in the last couple of years. Using several different in vivo and ex vivo optical imaging techniques, we here visualized and quantified the benefit of RGD- and NGR-based vascular vs EPR-mediated passive tumor targeting. This was done using ∼ 10 nm sized polymeric nanocarriers, which were either labeled with DY-676 (peptide-modified polymers) or with DY-750 (peptide-free polymers). Upon coinjection into mice bearing both highly leaky CT26 and poorly leaky BxPC3 tumors, it was found that vascular targeting did work, resulting in rapid and efficient early binding to tumor blood vessels, but that over time, passive targeting was significantly more efficient, leading to higher overall levels and to more efficient retention within tumors. Although this situation might be different for larger carrier materials, these insights indicate that caution should be taken not to overestimate the potential of active over passive tumor targeting.


Assuntos
Antineoplásicos/administração & dosagem , Nanocápsulas/química , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Oligopeptídeos/farmacocinética , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Difusão , Humanos , Camundongos , Nanocápsulas/administração & dosagem , Nanocápsulas/ultraestrutura , Nanocompostos/química , Nanocompostos/ultraestrutura , Neoplasias Experimentais/patologia , Oligopeptídeos/química , Tamanho da Partícula
10.
Biomacromolecules ; 15(7): 2590-9, 2014 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-24857680

RESUMO

Coiled coils are a common structural motif in many natural proteins that can also be utilized in the design and preparation of drug delivery systems for the noncovalent connection of two macromolecules. In this work, two different pairs of peptides forming coiled coil hetero-oligomers were designed, synthesized, and characterized. While the peptide sequences (VAALEKE)4 and (VAALKEK)4 predominantly form coiled coil heterodimers with randomly orientated peptide chains, (IAALESE)2-IAALESKIAALESE and IAALKSKIAALKSE-(IAALKSK)2 tend to form higher hetero-oligomers with an antiparallel orientation of their peptide chains. The associative behavior of these peptides was studied in aqueous solutions using circular dichroism spectroscopy, size-exclusion chromatography, isothermal titration calorimetry and sedimentation analyses. The orientation of the peptide chains in the coiled coil heterodimers was assessed using fluorescence spectroscopy with fluorescence resonance energy transfer labels attached to the ends of the peptides. The formation of the heterodimer can be used as a general method for the selective noncovalent conjugation of a specific targeting moiety with various drug carrier systems; this process involves simple self-assembly in a physiological solution before drug administration. The preparation of targeted macromolecular therapeutics consisting of a synthetic polymer drug carrier and a recombinant protein targeting ligand is discussed.


Assuntos
Portadores de Fármacos/química , Metacrilatos/química , Oligopeptídeos/química , Sequência de Aminoácidos , Estrutura Secundária de Proteína , Técnicas de Síntese em Fase Sólida
11.
Biomacromolecules ; 14(3): 881-9, 2013 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-23373696

RESUMO

The specificity of polymer conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) bearing cytostatic drugs for cancer cells could be significantly increased by the incorporation of a suitable targeting ligand, such as a monoclonal antibody (mAb). However, direct binding of the protein to the polymer carrier could cause considerable problems, such as decreasing the binding capacity of mAb to its target. Here, we introduce a novel strategy of joining a targeting moiety to a polymeric conjugate with cytostatic drug. The scFv of B1 mAb (specific for BCL1 leukemia cells) was tagged with peptide K ((VAALKEK)4). Peptide E ((VAALEKE)4), which forms a stable coiled coil structure heterodimer with peptide K, was assembled with the HPMA copolymers bearing doxorubicin. Such targeted polymeric conjugates possess very selective and high binding activity toward BCL1 cells. Similarly, targeted polymeric conjugates exert approximately 100 times higher cytostatic activity toward BCL1 cells in comparison to nontargeted conjugates in vitro. At the same time, the conjugates have comparable and rather low cytostatic activity for 38C13 cells, which are used as a negative control, in vitro.


Assuntos
Acrilamidas/farmacologia , Materiais Biocompatíveis/farmacologia , Citostáticos/farmacologia , Leucemia/tratamento farmacológico , Polímeros/farmacologia , Acrilamidas/síntese química , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Anticorpos Monoclonais/química , Materiais Biocompatíveis/síntese química , Linhagem Celular Tumoral , Proliferação de Células , Citostáticos/química , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Metacrilatos/química , Camundongos , Camundongos Endogâmicos BALB C , Polímeros/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
12.
Biomacromolecules ; 14(11): 4061-70, 2013 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-24083567

RESUMO

We report kinetic studies of therapeutically highly potent polymer-drug conjugates consisting of amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers bearing the anticancer drug doxorubicin (Dox). Highly hydrophobic cholesterol moieties as well as the drug were attached to the polymer backbone by a pH-sensitive hydrazone bond. Moreover, the structure of the spacer between the polymer carrier and the cholesterol moiety differed in order to influence the release rate of the hydrophobic moiety, and thus the disintegration of the high-molecular-weight micellar nanoparticle structure. We performed time-dependent SAXS/SANS measurements after changing pH from a typical blood value (pH 7.2) to that of tumor cells (pH 5.0) to characterize the drug release and changes in particle size and shape. Nanoparticles composed of the conjugates containing Dox were generally larger than the drug-free ones. For most conjugates, nanoparticle growth or decay was observed in the time range of several hours. It was established that the growth/decay rate and the steady-state size of nanoparticles depend on the spacer structure. From analytical fitting, we conclude that the most probable structure of the nanoparticles was a core-shell or a core with attached Gaussian chains. We concluded that the spacer structure determined the fate of a cholesterol derivative after the pH jump. Fitting results for 5α-cholestan-3-onecholestan-3-one and cholesteryl-4-oxopentanoate (Lev-chol) implied that cholesterol moieties continuously escape from the core of the nanoparticle core and concentrate in the hydrophilic shell. In contrast, cholest-4-en-3-one spacer prevent cholesterol escaping. Dox moiety release was only observed after a change in pH. Such findings justify the model proposed in our previous paper. Lastly, the cholesteryl 4-(2-oxopropyl)benzoate (Opb-Chol) was a different case where after the release of hydrophobic Opb-Chol moieties, the core becomes more compact. The physicochemical mechanisms responsible for the scenarios of the different spacers are discussed.


Assuntos
Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Micelas , Ácidos Polimetacrílicos/química , Acrilamidas/química , Colesterol/química , Concentração de Íons de Hidrogênio , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Cinética , Estrutura Molecular , Difração de Nêutrons , Tamanho da Partícula , Espalhamento a Baixo Ângulo , Propriedades de Superfície , Fatores de Tempo , Difração de Raios X
13.
Bioorg Med Chem ; 21(18): 5669-72, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23941688

RESUMO

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 24h in a pH 5.0 phosphate saline buffer. The ellipticinium drug retained the antiproliferative activity of the ellipticine.


Assuntos
Acrilamidas/química , Antineoplásicos/química , Portadores de Fármacos/química , Elipticinas/química , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , DNA/química , DNA/metabolismo , Portadores de Fármacos/síntese química , Humanos , Hidrazonas/química , Concentração de Íons de Hidrogênio
14.
Biomacromolecules ; 13(3): 652-63, 2012 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-22263698

RESUMO

Preclinical in vivo characterization of new polymeric drug conjugate candidates is crucial for understanding the effects of certain chemical modifications on distribution and elimination of these carrier systems, which is the basis for rational drug design. In our study we synthesized dual fluorescent HPMA copolymers of different architectures and molecular weights, containing one fluorescent dye coupled via a stable hydrazide bond functioning as the carrier label and the other one modeling the drug bound to a carrier via a pH-sensitive hydrolytically cleavable hydrazone bond. Thus, it was possible to track the in vivo fate, namely distribution, elimination and tumor accumulation, of the polymer drug carrier and a cleavable model drug simultaneously and noninvasively in nude mice using multispectral optical imaging. We confirmed our in vivo results by more detailed ex vivo characterization (imaging and microscopy) of autopsied organs and tumors. There was no significant difference in relative biodistribution in the body between the 30 KDa linear and 200 KDa star-like polymer, but the star-like polymer circulated much longer. We observed a moderate accumulation of the polymeric carriers in the tumors. The accumulation of the pH-sensitive releasable model drug was even higher compared to the polymer accumulation. Additionally, we were able to follow the long-term in vivo fate and to prove a time-dependent tumor accumulation of HPMA copolymers over several days.


Assuntos
Antineoplásicos/farmacologia , Carbocianinas/farmacologia , Neoplasias do Colo/tratamento farmacológico , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Indóis/farmacologia , Metacrilatos/química , Polímeros/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Benzopiranos , Carbocianinas/química , Carbocianinas/farmacocinética , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Preparações de Ação Retardada , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Indóis/química , Indóis/farmacocinética , Camundongos , Camundongos Nus , Polímeros/química , Polímeros/farmacocinética , Distribuição Tecidual
15.
Biomacromolecules ; 13(8): 2594-604, 2012 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-22793269

RESUMO

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.


Assuntos
Acrilamidas/química , Antibióticos Antineoplásicos/química , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Nanocápsulas/química , Algoritmos , Anisotropia , Colesterol , Interações Hidrofóbicas e Hidrofílicas , Luz , Substâncias Macromoleculares/química , Micelas , Modelos Moleculares , Conformação Molecular , Difração de Nêutrons , Tamanho da Partícula , Espalhamento a Baixo Ângulo , Difração de Raios X
16.
Bioorg Med Chem ; 20(13): 4056-63, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22658535

RESUMO

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.


Assuntos
Amsacrina/análogos & derivados , Antineoplásicos/química , Portadores de Fármacos/química , Polímeros/química , Amsacrina/química , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Portadores de Fármacos/síntese química , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Substâncias Intercalantes/síntese química , Substâncias Intercalantes/química , Substâncias Intercalantes/toxicidade , Microscopia de Fluorescência , Polímeros/síntese química , Água/química
17.
Int J Cancer ; 129(8): 2002-12, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21165950

RESUMO

Interleukin (IL)-2 has been approved for treatment of metastatic renal cancer and malignant melanoma. However, its unfavorable pharmacologic properties, severe side effects and the negative role of IL-2 in maintaining T regulatory cells are severe drawbacks. It has been shown that immunocomplexes of IL-2 and certain anti-IL-2 mAbs possess selective and high stimulatory activity in vivo. Here, we show that IL-2/S4B6 mAb immunocomplexes expand not only CD122(high) subsets and newly activated CD8(+) T cells but also natural killer T cells and γδ T cells. Further, we demonstrate that natural killer (NK) cells expanded by IL-2/S4B6 mAb immunocomplexes in vivo have high cytolytic activity, which can be further increased by coadministration of IL-12. We also demonstrate that IL-2/S4B6 mAb immunocomplexes possess noticeable antitumor activity in two syngeneic mouse tumor models, namely BCL1 leukemia and B16F10 melanoma, but only if administered early in tumor progression. To effectively treat established tumors, we administered the tumor-bearing mice first with N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugate, and subsequently with IL-2/S4B6 mAb immunocomplexes alone or with IL-12 to induce an efficient antitumor immune response. Importantly, we show that the conjugate has significantly lower immunosuppressive activity than free doxorubicin when using dosage with comparable antitumor activity, thus eliminating the majority of tumor cells while leaving the immune system mostly unimpaired for stimulation with IL-2/S4B6 mAb immunocomplexes. Indeed, we demonstrate that the conjugate and IL-2/S4B6 mAb immunocomplexes together have synergistic antitumor activity.


Assuntos
Acrilamidas/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Doxorrubicina/administração & dosagem , Imunoconjugados/uso terapêutico , Imunossupressores/administração & dosagem , Interleucina-2/imunologia , Interleucina-2/uso terapêutico , Células Matadoras Naturais/imunologia , Leucemia Experimental/terapia , Melanoma Experimental/terapia , Animais , Linfócitos T CD8-Positivos/imunologia , Feminino , Interleucina-12/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
18.
Anal Chem ; 83(13): 5458-62, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21634803

RESUMO

Traditional tissue-sectioning techniques for histological samples utilize various embedding media to stabilize the tissue on a sectioning target and to provide a smooth cutting surface. Due to the ion suppression effect in MALDI ionization and number of background peaks in the low-mass region, these media are not suitable for mass spectrometry imaging (MSI) experiments. To overcome this, droplets of water are often used to mount the tissue on a sectioning target, but the ice block formed around the tissue does not provide a good support for sectioning of fragile samples. In this work, we propose a novel embedding media, compatible with MALDI ionization and MSI experiments, based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). Using a reversible addition-fragmentation chain transfer polymerization technique, well-defined pHPMA polymer with narrow mass distribution was prepared. Benefits of the resulted pHPMA-based embedding media were tested on different tissue samples.


Assuntos
Acrilamidas/química , Polímeros/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Inclusão do Tecido , Animais , Abelhas , Pulmão , Camundongos
19.
Bioconjug Chem ; 22(6): 1194-201, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21513348

RESUMO

Radioactive decay of some radionuclides produces a shower of Auger electrons, potent ionizing radiation within a very short range in living tissue (typically ca. 100 nm). Therefore, they must be brought to DNA-containing cell compartments and preferentially directly to DNA to be fully biologically effective. They may be used for a triple-targeting approach (first targeting, polymer-based system targeting into tumor tissue due to EPR effect; second targeting, pH-controlled release of intercalator-bound Auger electron emitter in slightly acidic tumor tissue or endosome; third targeting, into DNA in cell nucleus by the intercalator) minimizing radiation burden of healthy tissues. We describe a first system of this type, an ellipticine derivative-bound iodine-125 attached to hydrazide moieties containing poly[N-(2-hydroxypropyl)methacrylamide]. The system is stable at pH 7.4 (0% intercalator released after 24 h incubation), while iodine-containing biologically active intercalator is released upon decrease of pH (25% intercalator released after 24 h incubation at pH 5.0-model of late endosomes). Both 2-N-(2-oxobutyl)-9-iodoellipticinium bromide and the noniodinated 2-N-(2-oxobutyl)ellipticinium bromide are potent intercalators, as proven by direct titration with DNA and ethidium displacement assay, and readily penetrate into cell nuclei, as proven by confocal microscopy. They retain chemotherapeutical antiproliferative properties of ellipticine against Raji, EL-4, and 4T1cells with IC(50) in the range 0.27-8.8 µmol/L. Polymer conjugate of 2-N-(2-oxobutyl)-9-iodoellipticinium bromide is internalized into endosomes, releases active drug, possesses cytotoxic activity, and the drug accumulates in cell nuclei.


Assuntos
Elétrons , Elipticinas/farmacologia , Organelas/química , Ácidos Polimetacrílicos/farmacologia , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , DNA/química , Relação Dose-Resposta a Droga , Elipticinas/química , Humanos , Hidrazinas/química , Concentração de Íons de Hidrogênio , Radioisótopos do Iodo , Camundongos , Estrutura Molecular , Organelas/efeitos dos fármacos , Ácidos Polimetacrílicos/síntese química , Ácidos Polimetacrílicos/química , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição Tecidual
20.
Bioconjug Chem ; 22(2): 169-79, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21218805

RESUMO

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.


Assuntos
Nanopartículas/química , Polímeros/química , RNA Interferente Pequeno/química , Dissulfetos/química , Estrutura Molecular , Polímeros/síntese química , Estereoisomerismo , Propriedades de Superfície , Tionas/química
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