Targeted nanogel conjugate for improved stability and cellular permeability of curcumin: synthesis, pharmacokinetics, and tumor growth inhibition.

Curcumin (CUR) is a unique natural compound with promising anticancer and anti-inflammatory activities. However, the therapeutic efficacy of curcumin was challenged in clinical trials, mostly due to its low bioavailability, rapid metabolism, and elimination. We designed a nanodrug form of curcumin, which makes it stable and substantially enhances cellular permeability and anticancer activity at standard oral administration. Curcumin was conjugated as an ester to cholesteryl-hyaluronic acid (CHA) nanogel that is capable of targeted delivery to CD44-expressing drug-resistant cancer cells. CHA-CUR nanogels demonstrated excellent solubility and sustained drug release in physiological conditions. It induced apoptosis in cancer cells, suppressing the expression of NF-κB, TNF-α, and COX-2 cellular targets similar to free curcumin. Pharmacokinetic/pharmacodynamic (PK/PD) studies also revealed improved circulation parameters of CHA-CUR at oral, i.p. and i.v. administration routes. CHA-CUR showed targeted tumor accumulation and effective tumor growth inhibition in human pancreatic adenocarcinoma MiaPaCa-2 and aggressive orthotropic murine mammary carcinoma 4T1 animal models. CHA-CUR treatment was well-tolerated and resulted in up to 13-fold tumor suppression, making this nanodrug a potential candidate for cancer prevention and therapeutic treatment.

Encapsulation of poorly soluble drugs in polymer-drug conjugates: effect of dual-drug nanoformulations on cancer therapy.

PURPOSE: Current cancer chemotherapy is gradually shifting to the application of drug combinations that prevent development of drug resistance. Many anticancer drugs have poor solubility and limited oral bioavailability. Using an innovative approach, we developed dual-drug nanoformulations of a polymeric nanogel conjugate with anticancer 5-FU nucleoside analog, floxuridine (FLOX), and the second anticancer drugs, paclitaxel (PCL), or a geldanamycin analog, 17-AAG, for combination therapy. METHODS: PCL or 17-AAG had been encapsulated in the cholesteryl-polyvinyl alcohol-floxuridine nanogel (CPVA-FLOX) by simple solution mixing and sonication. Dual nanodrugs formed particles with diameter 180 nm and either drug content (5-20%) that were stable and could be administered orally. Their cytotoxicity in human and mouse cancer cells was determined by MTT assay, and cellular target inhibition - by Western blot analysis. Tumor growth inhibition was evaluated using an orthotopic mouse mammary 4T1 cancer model. RESULTS: CPVA-FLOX was more potent than free drug in cancer models including drug-resistant ones; while dual nanodrugs demonstrated a significant synergy (CPVA-FLOX/PCL), or showed no significant synergy (CPVA-FLOX/17-AAG) compared to free drugs (PCL or 17-AAG). Dual nanodrug CPVA-FLOX/17-AAG effect on its cellular target (HSP70) was similar to 17-AAG alone. In animal model, however, both dual nanodrugs effectively inhibited tumor growth compared to CPVA-FLOX after oral administration. CONCLUSION: Oral dual-drug nanoformulations of poorly-soluble drugs proved to be a highly efficient combination anticancer therapy in preclinical studies.

Nano-NRTIs demonstrate low neurotoxicity and high antiviral activity against HIV infection in the brain.

Antiviral therapy using nucleoside reverse transcriptase inhibitors (NRTIs) is neurotoxic and has low efficiency in eradication of HIV-1 harbored in central nervous system (CNS). Previously, we reported that active 5'-triphosphates of NRTIs encapsulated in cationic nanogels (nano-NRTIs) suppress HIV-1 activity more efficiently than NRTIs and exhibit reduced mitochondrial toxicity [Vinogradov SV, Poluektova LY, Makarov E, Gerson T, Senanayake MT. Nano-NRTIs: efficient inhibitors of HIV type-1 in macrophages with a reduced mitochondrial toxicity. Antivir Chem Chemother. 2010; 21:1-14. Makarov E, Gerson T, Senanayake T, Poluektova LY, Vinogradov. Efficient suppression of Human Immunodeficiency Virus in Macrophages by Nano-NRTIs. Antiviral Res. 2010; 86(1):A38-9]. Here, we demonstrated low neurotoxicity and excellent antiviral activity of nano-NRTIs decorated with the peptide (AP) binding brain-specific apolipoprotein E receptor. Nano-NRTIs induced lower levels of apoptosis and formation of reactive oxygen species, a major cause of neuron death, than free NRTIs. Optimization of size, surface decoration with AP significantly increased brain accumulation of nano-NRTIs. The efficient CNS delivery of nano-NRTIs resulted in up to 10-fold suppression of retroviral activity and reduced virus-associated inflammation in humanized mouse model of HIV-1 infection in the brain. Our data provide proof of the advanced efficacy of nano-NRTIs as safer alternative of current antiviral drugs. FROM THE CLINICAL EDITOR: This team of investigators demonstrated low neurotoxicity and excellent anti-HIV activity of nano-nucleoside reverse transcriptase inhibitors decorated with the peptide (AP) binding brain-specific apolipoprotein E receptor, providing proof of enhanced efficacy and a safer alternative compared with current antiviral drugs.

Hyaluronic acid-based nanogel-drug conjugates with enhanced anticancer activity designed for the targeting of CD44-positive and drug-resistant tumors.

Many drug-resistant tumors and cancer stem cells (CSC) express elevated levels of CD44 receptor, a cellular glycoprotein binding hyaluronic acid (HA). Here, we report the synthesis of nanogel-drug conjugates based on membranotropic cholesteryl-HA (CHA) for efficient targeting and suppression of drug-resistant tumors. These conjugates significantly increased the bioavailability of poorly soluble drugs with previously reported activity against CSC, such as etoposide, salinomycin, and curcumin. The small nanogel particles (diameter 20-40 nm) with a hydrophobic core and high drug loads (up to 20%) formed after ultrasonication and demonstrated a sustained drug release following the hydrolysis of biodegradable ester linkage. Importantly, CHA-drug nanogels demonstrated 2-7 times higher cytotoxicity in CD44-expressing drug-resistant human breast and pancreatic adenocarcinoma cells compared to that of free drugs and nonmodified HA-drug conjugates. These nanogels were efficiently internalized via CD44 receptor-mediated endocytosis and simultaneous interaction with the cancer cell membrane. Anchoring by cholesterol moieties in the cellular membrane after nanogel unfolding evidently caused more efficient drug accumulation in cancer cells compared to that in nonmodified HA-drug conjugates. CHA-drug nanogels were able to penetrate multicellular cancer spheroids and displayed a higher cytotoxic effect in the system modeling tumor environment than both free drugs and HA-drug conjugates. In conclusion, the proposed design of nanogel-drug conjugates allowed us to significantly enhance drug bioavailability, cancer cell targeting, and the treatment efficacy against drug-resistant cancer cells and multicellular spheroids.

Novel anticancer polymeric conjugates of activated nucleoside analogues.

Inherent or therapy-induced drug resistance is a major clinical setback in cancer treatment. The extensive usage of cytotoxic nucleobases and nucleoside analogues in chemotherapy also results in the development of specific mechanisms of drug resistance, such as nucleoside transport or activation deficiencies. These drugs are prodrugs; and being converted into the active mono-, di-, and triphosphates inside cancer cells following administration, they affect nucleic acid synthesis, nucleotide metabolism, or sensitivity to apoptosis. Previously, we actively promoted the idea that the nanodelivery of active nucleotide species, e.g., 5'-triphosphates of nucleoside analogues, can enhance drug efficacy and reduce nonspecific toxicity. In this study, we report the development of a novel type of drug nanoformulations, polymeric conjugates of nucleoside analogues, which are capable of the efficient transport and sustained release of phosphorylated drugs. These drug conjugates have been synthesized, starting from cholesterol-modified mucoadhesive polyvinyl alcohol or biodegradable dextrin, by covalent attachment of nucleoside analogues through a tetraphosphate linker. Association of cholesterol moieties in aqueous media resulted in intramolecular polymer folding and the formation of small nanogel particles containing 0.5 mmol/g of a 5'-phosphorylated nucleoside analogue, e.g., 5-fluoro-2'-deoxyuridine (floxuridine, FdU), an active metabolite of anticancer drug 5-fluorouracyl (5-FU). The polymeric conjugates demonstrated rapid enzymatic release of floxuridine 5'-phosphate and much slower drug release under hydrolytic conditions (pH 1.0-7.4). Among the panel of cancer cell lines, all studied polymeric FdU-conjugates demonstrated an up to 50× increased cytotoxicity in human prostate cancer PC-3, breast cancer MCF-7, and MDA-MB-231 cells, and more than 100× higher efficacy against cytarabine-resistant human T-lymphoma (CEM/araC/8) and gemcitabine-resistant follicular lymphoma (RL7/G) cells as compared to free drugs. In the initial in vivo screening, both PC-3 and RL7/G subcutaneous tumor xenograft models showed enhanced sensitivity to sustained drug release from polymeric FdU-conjugate after peritumoral injections and significant tumor growth inhibition. All these data demonstrate a remarkable clinical potential of novel polymeric conjugates of phosphorylated nucleoside analogues, especially as new therapeutic agents against drug-resistant tumors.

Multifunctional peptide-PEG intercalating conjugates: programmatic of gene delivery to the blood-brain barrier.

PURPOSE: To enhance transfection efficacy of pDNA through the application of multifunctional peptide-PEG-tris-acridine conjugates (pPAC) and the formation of biodegradable core-shell polyplexes for gene delivery to the blood-brain barrier (BBB). METHODS: pPAC-mediated transfection was compositionally optimized in mouse BBB cells (bEnd.3). Cellular uptake and trafficking, and brain accumulation of pDNA was evaluated by fluorescent imaging and histochemistry. We constructed anti-MRP4 siRNA-producing vectors and evaluated the efficacy of MRP4 down-regulation of MRP4 by Western blot and qPCR, and its effect on the uptake of (3)H-AZT, an MRP4 substrate. RESULTS: A core-shell gene delivery system (GDS) was assembled from pDNA and pPAC, carrying multifunctional peptides with NLS, TAT, and brain-specific BH, or ApoE sequences, and biodegradable pLPEI polyamine. This GDS demonstrated better cellular and nuclear accumulation, and a 25-fold higher transfection efficacy in slow-dividing bEnd.3 cells compared to ExGen500. Inclusion of brain-targeting pPAC enhanced in vivo accumulation of functional pDNA in brain capillaries. Treatment by encapsulated anti-MRP4 siRNA-producing pDNA caused transient down-regulation of MRP4, and, after intravenous injection in Balb/c mice, enhanced AZT uptake in the brain by 230-270%. CONCLUSIONS: The pPAC represent novel efficient components of GDS that could find various gene therapy applications, including genetic modulation of the BBB.

Efficient transfection of blood-brain barrier endothelial cells by lipoplexes and polyplexes in the presence of nuclear targeting NLS-PEG-acridine conjugates.

Brain capillary endothelial cells of the blood-brain barrier (BBB) are difficult targets for nonviral transfection even for the most potent transfection agents. Efficient protection and nuclear delivery of plasmid DNA are the key requirements for enhancing the transfection. We designed novel DNA intercalating conjugates of PEG-tris(acridine) with a short nuclear localization signal (NLS) peptide and investigated the effect of their complexes with luciferase-encoded plasmid DNA on lipoplex- and polyplex-mediated transfection of murine brain capillary endothelial bEnd.3 cells. These intercalation complexes protected DNA from nucleolytic degradation forming a protective PEG layer around plasmid DNA and could be efficiently condensed by Lipofectamine2000 or Exgen500 into nanosized particles. Complexation of plasmid DNA with a PEG-acridine/NLS-PEG-acridine mixture (9:1 w/w), taken in an amount equal to 5-6 NLS peptides per DNA molecule, significantly enhanced both lipo- and polyplex transfection efficacies and increased the number of transfected bEnd.3 endothelial cells in the presence of serum. Comparative transgene expression efficiency was significantly higher at longer PEG linker and optimal conjugate-to-DNA weight ratio, especially, at lower N/P ratio for both transfection agents, reaching 15-16-fold for lipoplexes and 10-11-fold for polyplexes. In addition, the NLS-PEG-acridine conjugates did not increase cytotoxicity of lipoplexes and polyplexes to bEnd.3 cells. These conjugates can serve as promising components for development of systemic nonviral transfecting approach to the transfection of the BBB and temporary modulation of its drug permeability.

Nanogels as pharmaceutical carriers: finite networks of infinite capabilities.

Nanogels are swollen nanosized networks composed of hydrophilic or amphiphilic polymer chains. They are developed as carriers for the transport of drugs, and can be designed to spontaneously incorporate biologically active molecules through formation of salt bonds, hydrogen bonds, or hydrophobic interactions. Polyelectrolyte nanogels can readily incorporate oppositely charged low-molecular-mass drugs and biomacromolecules such as oligo- and polynucleotides (siRNA, DNA) as well as proteins. The guest molecules interact electrostatically with the ionic polymer chains of the gel and become bound within the finite nanogel. Multiple chemical functionalities can be employed in the nanogels to introduce imaging labels and to allow targeted drug delivery. The latter can be achieved, for example, with degradable or cleavable cross-links. Recent studies suggest that nanogels have a very promising future in biomedical applications.

Polymeric nanogels containing the triphosphate form of cytotoxic nucleoside analogues show antitumor activity against breast and colorectal cancer cell lines.

The therapeutic efficiency of anticancer nucleoside analogues (NA) strongly depends on their intracellular accumulation and conversion into 5'-triphosphates. Because active NATP cannot be directly administrated due to instability, we present here a strategy of nanoencapsulation of these active drugs for efficient delivery to tumors. Stable lyophilized formulations of 5'-triphosphates of cytarabine (araCTP), gemcitabine (dFdCTP), and floxuridine (FdUTP) encapsulated in biodegradable PEG-cl-PEI or F127-cl-PEI nanogel networks (NGC and NGM, respectively) were prepared by a self-assembly procedure. Cellular penetration, in vitro cytotoxicity, and drug-induced cell cycle perturbations of these nanoformulations were analyzed in breast and colorectal cancer cell lines. Cellular accumulation and NATP release from nanogel was studied by confocal microscopy and direct high-performance liquid chromatography analysis of cellular lysates. Antiproliferative effect of dFdCTP nanoformulations was evaluated in human breast carcinoma MCF7 xenograft animal model. Nanoencapsulated araCTP, dFdCTP, and FdUTP showed similar to NA cytotoxicity and cell cycle perturbations. Nanogels without drugs showed very low cytotoxicity, although NGM was more toxic than NGC. Treatment by NATP nanoformulations induced fast increase of free intracellular drug concentration. In human breast carcinoma MCF7 xenograft animal model, i.v. dFdCTP-nanogel was equally effective in inhibiting tumor growth at four times lower administered drug dose compared with free gemcitabine. Active triphosphates of NA encapsulated in nanogels exhibit similar cytotoxicity and cell cycle perturbations in vitro and faster cell accumulation and equal tumor growth-inhibitory activity in vivo at much lower dose compared with parental drugs, illustrating their therapeutic potential for cancer chemotherapy.

Polymeric nanogel formulations of nucleoside analogs.

Nanogels are colloidal microgel carriers that have been recently introduced as a prospective drug delivery system for nucleotide therapeutics. The crosslinked protonated polymer network of nanogels binds oppositely charged drug molecules, encapsulating them into submicron particles with a core-shell structure. The nanogel network also provides a suitable template for chemical engineering, surface modification and vectorisation. This review reveals recent attempts to develop novel drug formulations of nanogels with antiviral and antiproliferative nucleoside analogs in the active form of 5'-triphosphates, discusses structural approaches to the optimisation of nanogel properties, and discusses the development of targeted nanogel drug formulations for systemic administration. Notably, nanogels can improve the CNS penetration of nucleoside analogs that are otherwise restricted from passing across the blood-brain barrier. The latest findings reviewed here demonstrate an efficient intracellular release of nucleoside analogs, encouraging further applications of nanogel carriers for targeted drug delivery.

Polyplex Nanogel formulations for drug delivery of cytotoxic nucleoside analogs.

Hydrophilic nanosized particles consisting of the cross-linked cationic polymer network (Nanogels) are suggested as a drug delivery system for nucleoside analog 5'-triphosphates, an active form of cytotoxic anticancer drugs. Preparation, properties, and cellular effects of several polyplex Nanogel formulations with the 5'-triphosphate of cytotoxic 5-fluoroadenine arabinoside (fludarabine) (FATP) were examined and discussed here. The polyplexes have formed spontaneously by mixing solutions of FATP and Nanogels because of ionic interactions between protonated polyethylenimine (PEI) chains in Nanogel network with polyphosphate groups of the drug. Subsequent compaction of the flexible Nanogel network has resulted in an encapsulation of the FATP/PEI complex in a dense core surrounded by hydrophilic poly(ethylene glycol) (PEG) envelope. This structure has provided a sustained release of the drug, as well as an efficient protection of FATP against enzymatic degradation. The drug loading could reach up to 33% by weight of the drug-Nanogel formulation. In vitro 35% of loaded drug has released from Nanogel formulations during the first 24 h, and a slower additional release was observed during the next 2 days. Nanogels have protected 90% of the encapsulated FATP from enzymatic dephosphorylation during the first 60 min of incubation in vitro. The drug-Nanogel formulation compared to the drug has demonstrated a significantly enhanced cytotoxicity in cultured cancer cells. Cancer cell-targeting molecules, such as folate, could be easily attached to Nanogels and this modification has resulted in a strong 10-fold increase of the carrier's internalization in human breast carcinoma MCF-7 cells. Moreover, transcellular transport of the folate-Nanogel polyplexes was found to be 4 times more effective compared to the drug alone using Caco-2 cell monolayers as an in vitro intestinal model. The data demonstrate that this carrier-based approach to delivery of cytotoxic drugs may enhance tumor specificity and significantly reduce side effects related to systemic toxicity usually observed during cancer chemotherapy.

Curcumin-encapsulating Nanogels as an Effective Anticancer Formulation for Intracellular Uptake.

Nanoscale drug delivery systems represent an attractive strategy to improve both the efficacy and safety of anticancer drugs. In this work, we describe nanoformulation of curcumin, a most potent natural anticancer compound capable of killing cancer cells while sparing the normal tissues. Since curcumin is a natural hydrophobic polyphenol, it has a low aqueous solubility and bioavailability, which are challenging to its therapeutic efficacy. We developed and evaluated a novel colloidal nanogel carrier for encapsulation of curcumin to increase its solubility and cytotoxicity. Amphiphilic Poloxamer-cationic network in the nanogel NG127 was designed to efficiently encapsulate curcumin. Homogenous drug complexes were obtained with 20-25% content of curcumin and the particle size of ca. 150 nm. Using ImageStream multispectral imaging flow cytometry, we demonstrated that the curcumin-nanogel formulation (C-NG) was readily internalized into MDA-231 breast cancer cells. A real-time cell growth electronic sensing assay was used to measure proliferation responses of various breast cancer cells to C-NG treatments. Our results indicated that the C-NG formulation was 70-85% more effective in inhibiting growth, at concentrations lower than IC50 of free curcumin. This was also confirmed morphologically by modified acridine orange/ethidium bromide staining and fluorescent microscopy. Importantly, nanocarrier NG127 alone displayed practically no cytotoxicity. We conclude that nanogel carriers offer an innovative way to encapsulate curcumin and to obtain more effective anticancer therapeutics than curcumin alone with a potential to specific tumor targeting, such as using antibodies against surface receptors specific to breast cancer cells.

Nanosized cationic hydrogels for drug delivery: preparation, properties and interactions with cells.

A new family of nanoscale materials on the basis of dispersed networks of cross-linked ionic and nonionic hydrophilic polymers is being developed. One example is the nanosized cationic network of cross-linked poly(ethylene oxide) (PEO) and polyethyleneimine (PEI), PEO-cl-PEI nanogel. Interaction of anionic amphiphilic molecules or oligonucleotides with PEO-cl-PEI results in formation of nanocomposite materials in which the hydrophobic regions from polyion-complexes are joined by the hydrophilic PEO chains. Formation of polyion-complexes leads to the collapse of the dispersed gel particles. However, the complexes form stable aqueous dispersions due to the stabilizing effect of the PEO chain. These systems allow for immobilization of negatively charged biologically active compounds such as retinoic acid, indomethacin and oligonucleotides (bound to polycation chains) or hydrophobic molecules (incorporated into nonpolar regions of polyion-surfactant complexes). The nanogel particles carrying biological active compounds have been modified with polypeptide ligands to enhance receptor-mediated delivery. Efficient cellular uptake and intracellular release of oligonucleotides immobilized in PEO-cl-PEI nanogel have been demonstrated. Antisense activity of an oligonucleotide in a cell model was elevated as a result of formulation of oligonucleotide with the nanogel. This delivery system has a potential of enhancing oral and brain bioavailability of oligonucleotides as demonstrated using polarized epithelial and brain microvessel endothelial cell monolayers.

Mixed polymer micelles of amphiphilic and cationic copolymers for delivery of antisense oligonucleotides.

Cationic copolymers were synthesized by conjugation of branched 2 kDa polyethylenimine (PEI) and Pluronic block copolymers (F38, P85, P123). Compositions of these copolymers mixed with corresponding free Pluronics at weight ratio 1:9 were used to complex phosphorothioate oligonucleotides (ODN). As a result stable suspensions of small micelle-like particles (<220 nm) were obtained. Incorporation of ODN in these formulations increased uptake of ODN in KBv cells and increased sequence specific activity of antisense ODN targeted against MDR gene in multidrug resistant cells resulting in inhibition of the functional activity of P-glycoprotein (P-gp) in these cells. Furthermore, these formulations increased transport of ODN across model intestinal barrier, Caco-2 cell monolayers, suggesting that they could be useful for oral delivery of biologically active ODN.

Application of activated nucleoside analogs for the treatment of drug-resistant tumors by oral delivery of nanogel-drug conjugates.

A majority of nanoencapsulated drugs that have shown promise in cancer chemotherapy are administered intravenously. Development of effective oral nanoformulations presents a very challenging medical goal. Here, we describe successful applications of innovative polymeric nanogels in the form of conjugates with activated nucleoside analogs for oral administration in cancer chemotherapy. Previously, we reported the synthesis of amphiphilic polyvinyl alcohol and dextrin-based nanogel conjugates with the phosphorylated 5-FU nucleoside Floxuridine and demonstrated their enhanced activity against regular and drug-resistant cancers (T.H. Senanayake, G. Warren, S.V. Vinogradov, Novel anticancer polymeric conjugates of activated nucleoside analogs, Bioconjug. Chem. 22 (2011) 1983-1993). In this study, we synthesized and evaluated oral applications of nanogel conjugates of a protected Gemcitabine, the drug never used in oral therapies. These conjugates were able to quickly release an active form of the drug (Gemcitabine 5'-mono-, di- and triphosphates) by specific enzymatic activities, or slowly during hydrolysis. Gemcitabine conjugates demonstrated up to 127 times higher in vitro efficacy than the free drug against various cancer cells, including the lines resistant to nucleoside analogs. Surprisingly, these nanogel-drug conjugates were relatively stable in gastric conditions and able to actively penetrate through the gastrointestinal barrier based on permeability studies in Caco-2 cell model. In tumor xenograft models of several drug-resistant human cancers, we observed an efficient inhibition of tumor growth and extended the life-span of the animals by 3 times that of the control with orally treated Gemcitabine- or Floxuridine-nanogel conjugates. Thus, we have demonstrated a potential of therapeutic nanogel conjugates with the activated and stabilized Gemcitabine as a successful oral drug form against Gemcitabine-resistant and other drug-resistant tumors.

The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC.

Short biodegradable polyamines for gene delivery and transfection of brain capillary endothelial cells.

With the goal of rational design of systemic gene delivery system and efficient transfection of capillary endothelial cells forming the blood-brain barrier (BBB), we synthesized several short polyamines with reducible disulfide backbones for pDNA packaging, internalization and consequent release from endosomal compartments. The synthetic cationic polymers prepared from short linear PEI (pLPEI), triethylenetetramine (pTETA), and spermine (pSPE), demonstrated very low toxicity, good condensation capacity, and high levels of pDNA protection, producing small particulate nanoformulations. Mild reduction of the disulfide backbone allowed complete release of pDNA from these polyplexes. In vitro transfection of murine brain capillary endothelial bEnd.3 cells with pSPE, pTETA, and pLPEI polyplexes was 2.3-4.9 times more effective compared with the non-degradable LPEI 22kDa reagent (ExGen500) in the presence of serum. Their transfection ability was noticeably decreased following inhibition of the cellular reduced glutathione (GSH). After cellular uptake of biodegradable polyplexes, a disperse distribution of labeled pDNA in the cytoplasm of transfected cells was observed in contrast to ExGen500. Based on these polyamines, novel multifunctional polyplexes have been developed for efficient nuclear delivery of pDNA by co-application of NLS peptide and PEG-modified intercalating conjugates. Significant increase of nuclear accumulation was observed, and the transfection of bEnd.3 cells was additionally enhanced nearly 2-fold, demonstrating 8.5-, 6.3- and 3.7-fold better levels for pLPEI, pTETA, and pSPE, respectively, compared to ExGen500. Following brain-specific targeting, these safe and effective polyplexes may be converted into systemic nanocarriers for gene delivery and transfection of the BBB.

Efficient overcoming of drug resistance to anticancer nucleoside analogs by nanodelivery of active phosphorylated drugs.

One of the major problems in cancer chemotherapy is the fast development of drug resistance to most anticancer therapeutics. Thus, an important cause of the eventual decline in clinical efficacy of cytotoxic nucleoside analogs was the selection of resistant cancer cells with deficiencies in the expression of nucleoside transporters or nucleoside-activating kinases. Here, we present an efficient strategy of overcoming this type of drug resistance by tumor-specific delivery of nanogel-encapsulated active triphosphates of nucleoside analogs (NATP). The small particles of biodegradable cationic nanogels loaded with anionic NATP efficiently interacted with cancer cells and released active drug compounds into the cytoplasm. The potential of novel drug formulations was evaluated in the nucleoside transport-deficient (CEM/araC/C8) or nucleoside activation-deficient (RL7/G) lymphogenic cancer cells. Compared to nucleoside analogs, NATP-loaded nanogels demonstrated increased cytotoxicity, reducing the drug resistance index 250- to 900-fold in CEM/araC/C8 cells and 70- to 100-fold in RL7/G cells. The strong cytotoxic effect of nanoformulations was accompanied by characteristic cell cycle perturbations, usually observed in drug-treated sensitive cells, and resulted in the induction of apoptosis in all studied drug-resistant cells. Efficient cellular accumulation of nanogels and the consequent increase in intracellular levels of NATP were found to be the major factors determining cytotoxic efficacy of nanoformulations. Decoration of nanogels with multiple molecules of tumor lymphatic-specific peptide (LyP1) enhanced the binding efficacy of nanocarriers with lymphogenic cancer cells. The targeted nanoformulation of activated gemcitabine (LyP1-NG-dFdCTP), when injected in subcutaneous RL7/G xenograft tumor model, demonstrated 2-fold more efficient tumor growth inhibition than gemcitabine at a higher dose. Nanogel-drug formulations exhibited no systemic toxicity during the treatment, hence extending the versatility of nucleoside analogs in the treatment of drug-resistant lymphogenic tumors.

Nano-NRTIs: efficient inhibitors of HIV type-1 in macrophages with a reduced mitochondrial toxicity.

BACKGROUND: Macrophages serve as a depot for HIV type-1 (HIV-1) in the central nervous system. To efficiently target macrophages, we developed nanocarriers for potential brain delivery of activated nucleoside reverse transcriptase inhibitors (NRTIs) called nano-NRTIs. METHODS: Nanogel carriers consisting of poly(ethylene glycol) (PEG)- or Pluronic-polyethylenimine (PEI) biodegradable networks, star PEG-PEI or poly(amidoamine) dendrimer-PEI-PEG dendritic networks, as well as nanogels decorated with brain-targeting peptide molecules, specifically binding to the apolipoprotein E receptor, were synthesized and evaluated. Nano-NRTIs were obtained by mixing aqueous solutions of zidovudine 5'-triphosphate or didanosine 5'-triphosphate and nanocarriers, followed by freeze-drying. Intracellular accumulation, cytotoxicity and antiviral activity of nano-NRTIs were monitored in monocyte-derived macrophages (MDMs). HIV-1 viral activity in infected MDMs was measured by a reverse transcriptase activity assay following treatment with nano-NRTIs. Mitochondrial DNA depletion in MDMs and human HepG2 cells was assessed by quantitative PCR. RESULTS: Nanogels were efficiently captured by MDMs and demonstrated low cytotoxicity, and no antiviral activity without drugs. All nano-NRTIs demonstrated high efficacy of HIV-1 inhibition at drug levels as low as 1 µmol/l, representing a 4.9- to 14-fold decrease in 90% effective drug concentrations as compared with NRTIs, whereas 50% cytotoxicity effects started at 200× higher concentrations. Nano-NRTIs with a core-shell structure and decorated with brain-targeting peptides displayed the highest antiviral efficacy. Mitochondrial DNA depletion, a major cause of NRTI neurotoxicity, was reduced threefold compared with NRTIs at application of selected nano-NRTIs. CONCLUSIONS: Nano-NRTIs demonstrated a promising antiviral efficacy against HIV-1 in MDMs and showed strong potential as nanocarriers for delivery of antiviral drugs to macrophages harbouring in the brain.