Nanocarriers in photodynamic therapy—in vitro and in vivo studies

Photodynamic therapy (PDT) is a minimally invasive technique which has proven to be successful in the treatment of several types of tumors. This relatively simple method exploits three inseparable elements: phototoxic compound (photosensitizer [PS]), light source, and oxygen. Upon irradiation by light with specified wavelength, PS generates reactive oxygen species, which starts the cascade of reactions leading to cell death. The positive therapeutic outcome of PDT may be limited due to several aspects, including low water solubility of PSs, hampering their effective administration and blood circulation, as well as low tumor specificity, inefficient cellular uptake and activation energies requiring prolonged illumination times. One of the promising approaches to overcome these obstacles involves the use of carrier systems modulating pharmacokinetics and pharmacodynamics of the PSs. In the present review, we summarized current in vitro and in vivo studies regarding the use of nanoparticles as potential delivery devices for PSs to enhance their cellular uptake and cytotoxic properties, and thus—the therapeutic outcome of PDT

Poly(propyleneimine) dendrimers as carriers of anticancer adenosine nucleotides

Pracę stanowi cykl publikacji.Leki z grupy analogów adenozynowych należą do antymetabolitów o szerokim zastosowaniu w leczeniu białaczek i chłoniaków. Związki te dla swojej aktywności cytotoksycznej wymagają ułatwionego transportu dokomórkowego za pośrednictwem specyficznych transporterów błonowych oraz fosforylacji przez kinazy wewnątrzkomórkowe, skutkującej tworzeniem aktywnych form trójfosforanowych. Tak złożony metabolizm może prowadzić do zmniejszenia efektu terapeutycznego i wystąpienia oporności komórkowej związanej z obniżeniem ekspresji transporterów lub aktywności wewnątrzkomórkowych kinaz. W celu pokonania tych ograniczeń i zwiększenia skuteczności terapii przeciwnowotworowej z wykorzystaniem analogów adenozynowych, zaproponowano bezpośredni dokomórkowy transport aktywnych, trójfosforanowych form leków za pomocą nanonośników. Liczne badania potwierdziły, że rozgałęzione dendrymery o ściśle określonej strukturze są dobrymi kandydatami na nośniki leków. Dowiedziono, że dendrymery mogą przenosić dużą liczbę cząsteczek aktywnych, jednocześnie zapewniając ich zwiększoną rozpuszczalność i kontrolowaną biodystrybucję. Ponadto niewielki rozmiar i kulisty kształt tych polimerów sprzyjają ich wnikaniu do komórek i długotrwałemu krążeniu we krwi. Głównym celem pracy była charakterystyka kompleksów nukleotydów adenozynowych z dendrymerami polipropylenoiminowymi generacji 4 (PPI G4) oraz ocena możliwości zastosowania ich w terapii przeciwnowotworowej. Wykazano, że dendrymery PPI mogą skutecznie oddziaływać z nukleotydami i tworzyć stabilne kompleksy poprzez oddziaływania elektrostatyczne między zjonizowanymi grupami fosforanowymi trójfosforanu i aminowymi dendrymeru. Proces ten silnie zależy od warunków środowiskowych, szczególnie pH i obecności jonów nieorganicznych. Dowiedziono, że dendrymer PPI generacji 4 o powierzchni częściowo zmodyfikowanej maltozą (PPI-Mal OS G4) może służyć jako efektywny nośnik dla trójfosforanowej formy fludarabiny, zwiększając jej dokomórkowy transport, a tym samym jej cytotoksyczność i pokonując oporność związaną z obniżoną ekspresją lub aktywnością hENT1 dzięki autonomicznej drodze wejścia do komórki. Z kolei potencjalnie silniejsze oddziaływania trójfosforanu klofarabiny z dendrymerami PPI i jego zdolność do maskowania dodatniego ładunku powierzchniowego tych polimerów może ograniczyć zdolność kompleksów do wewnątrzkomórkowego dostarczania i uwalniania aktywnej formy leku. Przeprowadzone badania wskazują na możliwość zastosowania dendrymerów PPI jako nośników dla adenozynowych leków nukleotydowych w celu zwiększenia ich stężenia wewnątrzkomórkowego oraz aktywności przeciwnowotworowej. Należy jednak podkreślić fakt, że stabilność i właściwości kompleksów nukleotyd-dendrymer silnie zależą od warunków środowiskowych, a także od struktury chemicznej leku, co należy wziąć pod uwagę podczas projektowania doświadczeń zarówno in vitro, jak i in vivo.Projekt OPUS „Komórkowe i molekularne mechanizmy działania kompleksów dendrymerów PPI z lekami przeciwnowotworowymi – analogami nukleozydowymi”, 2014/13/B/NZ3/04643; COST Action CA17140 „Nano2Clinic. Cancer Nanomedicine – from the bench to the bedside

Sugar Modification Enhances Cytotoxic Activity of PAMAM-Doxorubicin Conjugate in Glucose-Deprived MCF-7 Cells – Possible Role of GLUT1 Transporter

Purpose: In order to overcome the obstacles and side effects of classical chemotherapy, numerous studies have been performed to develop the treatment based on targeted transport of active compounds directly to the site of action. Since tumor cells are featured with intensified glucose metabolism, we set out to develop innovative, glucose-modified PAMAM dendrimer for the delivery of doxorubicin to breast cancer cells. Methods: PAMAM-dox-glc conjugate was synthesized and characterized by 1H NMR, FT-IR, size and zeta potential measurements. The drug release rate from conjugate was evaluated by dialysis under different pH conditions. The expression level of GLUT family receptors in cells cultured in full and glucose-deprived medium was evaluated by quantitative real-time RT-PCR and flow cytometry. The cytotoxicity of conjugate in presence or absence of GLUT1 inhibitors was determined by MTT assay. Results: We showed that PAMAM-dox-glc conjugate exhibits pH-dependent drug release and increased cytotoxic activity compared to free drug in cells cultured in medium without glucose. Further, we proved that these cells overexpress transporters of GLUT family. The toxic effect of conjugate was eliminated by the application of specific GLUT1 inhibitors. Conclusion: Our findings revealed that the glucose moiety plays a crucial role in the recognition of cells with high expression of GLUT receptors. By selectively blocking GLUT1 transporter we showed its importance for the cytotoxic activity of PAMAM-dox-glc conjugate. These results suggest that PAMAM-glucose formulations may constitute an efficient platform for the specific delivery of anticancer drugs to tumor cells overexpressing transporters of GLUT family. © 2019, The Author(s)

Nanoparticles for Directed Immunomodulation: Mannose-Functionalized Glycodendrimers Induce Interleukin-8 in Myeloid Cell Lines

New therapeutic strategies for personalized medicine need to involve innovative pharmaceutical tools, for example, modular nanoparticles designed for direct immunomodulatory properties. We synthesized mannose-functionalized poly(propyleneimine) glycodendrimers with a novel architecture, where freely accessible mannose moieties are presented on poly(ethylene glycol)-based linkers embedded within an open-shell maltose coating. This design enhanced glycodendrimer bioactivity and led to complex functional effects in myeloid cells, with specific induction of interleukin-8 expression by mannose glycodendrimers detected in HL-60 and THP-1 cells. We concentrated on explaining the molecular mechanism of this phenomenon, which turned out to be different in both investigated cell lines: in HL-60 cells, transcriptional activation via AP-1 binding to the promoter predominated, while in THP-1 cells (which initially expressed less IL-8), induction was mediated mainly by mRNA stabilization. The success of directed immunomodulation, with synthetic design guided by assumptions about mannose-modified dendrimers as exogenous regulators of pro-inflammatory chemokine levels, opens new possibilities for designing bioactive nanoparticles. © 2021 The Authors. Published by American Chemical Society

Cellular uptake of rose bengal is mediated by OATP1B1/1B3 transporters

Due to its fluorescent properties and high yield of singlet oxygen, rose bengal (RB) is one of the most promising photosensitizers for cancer treatment. However, the negative charge of RB molecule may significantly hamper its intracellular delivery by passive diffusion through the cell membrane. Thus, specific membrane protein transporters may be needed. The organic anion transporting polypeptides (OATPs) family are a well-characterized group of membrane protein transporters, responsible for cellular uptake of a number of drugs. To our knowledge, this is the first study that evaluates cellular transport of RB mediated by the OATP transporter family. First, electrified liquid-liquid interface, together with biophysical analysis and molecular dynamics simulations were used to characterize the interaction of RB with several models of a cellular membranes. These experiments proved that RB interacts only with the membrane’s surface, without spontaneously crossing the lipid bilayer. Evaluation of intracellular uptake of RB by flow cytometry and confocal microscopy showed significant differences in uptake between liver and intestinal cell line models differing in expression of OATP transporters. The use of specific pharmacological inhibitors of OATPs, together with Western blotting and in silico analysis, indicated that OATPs are crucial for cellular uptake of RB

Poly(lysine) Dendrimers Form Complexes with siRNA and Provide Its Effcient Uptake by Myeloid Cells: Model Studies for Therapeutic Nucleic Acid Delivery

The disruption of the cellular pathways of protein biosynthesis through the mechanism of RNA interference has been recognized as a tool of great diagnostic and therapeutic significance. However, in order to fully exploit the potential of this phenomenon, efficient and safe carriers capable of overcoming extra-and intracellular barriers and delivering siRNA to the target cells are needed. Recently, attention has focused on the possibility of the application of multifunctional nanoparticles, dendrimers, as potential delivery devices for siRNA. The aim of the present work was to evaluate the formation of dendriplexes using novel poly(lysine) dendrimers (containing lysine and arginine or histidine residues in their structure), and to verify the hypothesis that the use of these polymers may allow an efficient method of siRNA transfer into the cells in vitro to be obtained. The fluorescence polarization studies, as well as zeta potential and hydrodynamic diameter measurements were used to characterize the dendrimer:siRNA complexes. The cytotoxicity of dendrimers and dendriplexes was evaluated with the resazurin-based assay. Using the flow cytometry technique, the efficiency of siRNA transport to the myeloid cells was determined. This approach allowed us to determine the properties and optimal molar ratios of dendrimer:siRNA complexes, as well as to demonstrate that poly(lysine) dendrimers may serve as efficient carriers of genetic material, being much more effective than the commercially available transfection agent Lipofectamine 2000. This outcome provides the basis for further research on the application of poly(lysine) dendrimers as carriers for nucleic acids in the field of gene therapy. © 2020 by the authors

Silver Nanoparticles Surface-Modified with Carbosilane Dendrons as Carriers of Anticancer siRNA

Gene therapy is a promising approach in cancer treatment; however, current methods have a number of limitations mainly due to the difficulty in delivering therapeutic nucleic acids to their sites of action. The application of non-viral carriers based on nanomaterials aims at protecting genetic material from degradation and enabling its effective intracellular transport. We proposed the use of silver nanoparticles (AgNPs) surface-modified with carbosilane dendrons as carriers of anticancer siRNA (siBcl-xl). Using gel electrophoresis, zeta potential and hydrodynamic diameter measurements, as well as transmission electron microscopy, we characterized AgNP:siRNA complexes and demonstrated the stability of nucleic acid in complexes in the presence of RNase. Hemolytic properties of free silver nanoparticles and complexes, their effect on lymphocyte proliferation and cytotoxic activity on HeLa cells were also examined. Confocal microscopy proved the effective cellular uptake of complexes, indicating the possible use of this type of silver nanoparticles as carriers of genetic material in gene therapy. © 2020 by the authors. Licensee MDPI, Basel, Switzerland