Nanotechnology Approaches for the Delivery of Exogenous siRNA for HIV Therapy.

RNA interference (RNAi) is triggered by oligonucleotides that are about 21-23 nucleotides long and are capable of inducing the destruction of complementary mRNA. The RNAi technique has been successfully utilized to target HIV replication; however, the main limitation to the successful utilization of this technique in vivo is the inability of naked siRNA to cross the cell membrane by diffusion due to its strong anionic charge and large molecular weight. This review describes current nonviral nanotechnological approaches to deliver anti-HIV siRNAs for the treatment of HIV infection.

Optimization of the fabrication of novel stealth PLA-based nanoparticles by dispersion polymerization using D-optimal mixture design.

PURPOSE: Nanoparticle size is important in drug delivery. Clearance of nanoparticles by cells of the reticuloendothelial system has been reported to increase with increase in particle size. Further, nanoparticles should be small enough to avoid lung or spleen filtering effects. Endocytosis and accumulation in tumor tissue by the enhanced permeability and retention effect are also processes that are influenced by particle size. We present the results of studies designed to optimize cross-linked biodegradable stealth polymeric nanoparticles fabricated by dispersion polymerization. METHODS: Nanoparticles were fabricated using different amounts of macromonomer, initiators, crosslinking agent and stabilizer in a dioxane/DMSO/water solvent system. Confirmation of nanoparticle formation was by scanning electron microscopy (SEM). Particle size was measured by dynamic light scattering (DLS). D-optimal mixture statistical experimental design was used for the experimental runs, followed by model generation (Scheffe polynomial) and optimization with the aid of a computer software. Model verification was done by comparing particle size data of some suggested solutions to the predicted particle sizes. RESULTS AND CONCLUSION: Data showed that average particle sizes follow the same trend as predicted by the model. Negative terms in the model corresponding to the cross-linking agent and stabilizer indicate the important factors for minimizing particle size.

Recent Developments in Combination Immunotherapy with Other Therapies and Nanoparticle-Based Therapy for Triple-Negative Breast Cancer (TNBC).

Triple-negative breast cancer (TNBC), lacking specific receptors found in other breast cancer subtypes, poses significant treatment challenges due to limited therapeutic options. Therefore, it is necessary to develop novel treatment approaches for TNBC. In the last few decades, many attempts have been reported for alternative tools for TNBC treatment: immunotherapy, radiotherapy, targeted therapy, combination therapy, and nanotechnology-based therapy. Among them, combination therapy and nanotechnology-based therapy show the most promise for TNBC treatment. This review outlines recent advancements in these areas, highlighting the efficacy of combination therapy (immunotherapy paired with chemotherapy, targeted therapy, or radiotherapy) in both preclinical and clinical stages and nanotechnology-based therapies utilizing various nanoparticles loaded with anticancer agents, nucleic acids, immunotherapeutics, or CRISPRs in preclinical stages for TNBC treatment.

Triple Negative Breast Cancer Treatment Options and Limitations: Future Outlook.

Triple negative breast cancer (TNBC) has a negative expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptors (HER2). The survival rate for TNBC is generally worse than other breast cancer subtypes. TNBC treatment has made significant advances, but certain limitations remain. Treatment for TNBC can be challenging since the disease has various molecular subtypes. A variety of treatment options are available, such as chemotherapy, immunotherapy, radiotherapy, and surgery. Chemotherapy is the most common of these options. TNBC is generally treated with systemic chemotherapy using drugs such as anthracyclines and taxanes in neoadjuvant or adjuvant settings. Developing resistance to anticancer drugs and off-target toxicity are the primary hindrances to chemotherapeutic solutions for cancer. It is imperative that researchers, clinicians, and pharmaceutical companies work together to develop effective treatment options for TNBC. Several studies have suggested nanotechnology as a potential solution to the problem of suboptimal TNBC treatment. In this review, we summarized possible treatment options for TNBC, including chemotherapy, immunotherapy, targeted therapy, combination therapy, and nanoparticle-based therapy, and some solutions for the treatment of TNBC in the future. Moreover, we gave general information about TNBC in terms of its characteristics and aggressiveness.

Nanotechnology in the development of small and large molecule tyrosine kinase inhibitors and immunotherapy for the treatment of HER2-positive breast cancer.

The HER2 receptor tyrosine kinase is a member of the epidermal growth factor receptor family which includes EGFR, HER3 and HER4. They are known to play critical roles in both normal development and cancer. A subset of breast cancers is associated with the HER2 gene, which is amplified and/or overexpressed in 20-25% of invasive breast cancers and is correlated with tumor resistance to chemotherapy, Metastatic Breast Cancer (MBC) and poor patient survival. The advent of receptor tyrosine kinase inhibitors has improved the prognosis of HER2-postive breast cancers; however, HER2+MBC invariably progresses (acquired resistance or de novo resistance). The monoclonal antibody-based drugs (large molecule TKIs) target the extracellular binding domain of HER2; while the small molecule TKIs act intracellularly to inhibit proliferation and survival signals. We reviewed the modes of action of the TKIs with a view to showing which of the TKIs could be combined in nanoparticles to benefit from the power of nanotechnology (reduced toxicity, improved solubility of hydrophobic drugs, long circulation half-lives, circumventing efflux pumps and preventing capture by the reticuloendothelial system (mononuclear phagocyte system). Nanotherapeutics also mediate the synchronization of the pharmacokinetics and biodistribution of multiple drugs incorporated in the nanoparticles. Novel TKIs that are currently under investigation with or without nanoparticle delivery are mentioned, and nano-based strategies to improve their delivery are suggested. Immunotherapies currently in clinical practice, clinical trials or at the preclinical stage are discussed. However, immunotherapy only works well in relatively small subsets of patients. Combining nanomedicine with immunotherapy can boost therapeutic outcomes, by turning "cold" non-immunoresponsive tumors and metastases into "hot" immunoresponsive lesions.

Computer Optimization of Stealth Biodegradable Polymeric Dual-loaded Nanoparticles for Cancer Therapy Using Central Composite Face-centered Design.

BACKGROUND: Combination chemotherapy capable of overcoming cancer drug resistance can be facilitated by nanotechnology. OBJECTIVE: Synthesis, characterization, statistical experimental design, analysis and optimization of stealth pH-sensitive polymeric nanoparticles suitable as a platform for simultaneous delivery of paclitaxel and 17-AAG in breast cancer therapy were investigated. METHODS: An acetal crosslinker and a poly(ɛ)caprolactone macromonomer were synthesized and characterized. The statistical experimental design used was the response surface method (RSM). We used the central composite face-centered design (CCF) in three independent factors and seventeen runs. Nanoparticles were fabricated by dispersion polymerization techniques. Response variables evaluated were: particle size, drug loading, encapsulation efficiency, and in vitro availability. RESULTS: Scanning electron micrographs showed the formation of spherical nanoparticles. Computer software was used for the analysis of variance with a 95% confidence level and Q2 (goodness of prediction) to select an appropriate model for each of the response variables. Each term in each of the models was tested for the significance of the regression coefficients. The computer software optimizer was used for optimization to select factor combination to minimize particle size, time (h) for maximum release of paclitaxel and 17-AAG, to maximize paclitaxel and 17-AAG loading efficiency and to maximize paclitaxel and 17-AAG encapsulation efficiency. CONCLUSION: The optimization was successful, as shown by the validation data which lie within the confidence intervals of predicted values of the response variables. The selected factor combination is suitable for the in vivo evaluation of the nanoparticles loaded with paclitaxel and 17-AAG.

Studies on polyethylene glycol-monoclonal antibody conjugates for fabrication of nanoparticles for biomedical applications.

OBJECTIVE: The objective of this work is to synthesize and characterize PEGylated monoclonal antibody using the reactivity of oligosaccharide residues in the Fc region of trastuzumab and pertuzumab with a view to preserving their activities. METHODS: The hydrazide-functionalized PEG monomethacrylate was synthesized and reacted with NaIO4-generated aldehyde groups on glycans in the Fc-domain of trastuzumab and pertuzumab. The conjugates were purified by HPLC. SAMSA-fluorescein substitution method and MALDI MS spectroscopy were used to determine the number of PEG per antibody. Preliminary biological studies involved antiproliferative studies and binding (flow cytometry) following treatments with SKBR3 (HER2-overexpressing) cells and the control. RESULTS: 1H NMR and 13C NMR confirmed the formation of hydrazide-functionalized PEG monomethacrylate. MALDI mass-spectrometry showed that there are two PEGs per each antibody and it appears more reliable than the degree of SAMSA-fluorescein substitution method. HER-2 binding assay showed that PEGylated monoclonal antibody bound less efficiently to SKBR3 (high HER-2 expressing) cells than unmodified trastuzumab and pertuzumab. In vitro growth inhibitory effects of unmodified monoclonal antibodies increased with increase in concentration; while the in vitro growth inhibitory effects of PEGylated monoclonal antibodies also increased (but less than the pure antibody) with concentration and it appeared to be more active than unmodified mAbs at higher concentration. CONCLUSION: The results indicate that PEG can be site-specifically attached via the oxidized glycans in the Fc domain of monoclonal antibodies but the process needs further optimization in terms of PEG size and biological testing at each stage of development.

Drug Combinations in Breast Cancer Therapy.

Breast cancer therapy involves a multidisciplinary approach comprising surgery, radiotherapy, neoadjuvant and adjuvant therapy. Effective therapy of breast cancer requires maximum therapeutic efficacy, with minimal undesirable effects to ensure a good quality of life for patients. The carefully selected combination of therapeutic interventions provides patients with the opportunity to derive maximum benefit from therapy while minimizing or eliminating recurrence, resistance and toxic effects, as well as ensuring that patients have a good quality of life. This review discusses therapeutic options for breast cancer treatments and various combinations that had been previously exploited. The review will also give an insight into the potential application of the nanotechnology platform for codelivery of therapeutics in breast cancer therapy.

Disposition of naltrexone after intravenous bolus administration in Wistar rats, low-alcohol-drinking rats and high-alcohol-drinking rats.

Reports have shown that interspecies differences in the metabolism and pharmacokinetics of naltrexone are a rule rather than exception. However, there is paucity of information on the disposition of naltrexone in selectively bred rat lines that reliably exhibit high and low voluntary alcohol consumption, and are often used to study alcohol-drinking behavior. We have characterized the pharmacokinetic profiles of naltrexone in selectively bred rat lines: high-alcohol-drinking (HAD-1) and low-alcohol-drinking (LAD-1) rats as well as the native Wistar strain. This study was carried out to establish a baseline pharmacokinetic profile of naltrexone in these rats prior to evaluating its pharmacokinetic profile in polymeric controlled-release formulations in our laboratory. The hypothesis is that alcohol-preferring and non-alcohol-preferring lines of rats should differ in the disposition of intravenously administered naltrexone. Naltrexone administration and blood collection were via the jugular vein. In a parallel experiment, naltrexone was administered via the jugular vein, but urine was collected using the Nalgene metabolic cage system. Data were analyzed by a noncompartmental approach. Results show a high clearance that is close to or higher than hepatic blood flow in all groups (Wistar > LAD-1 > HAD-1, but with a statistically significant difference only between Wistar and HAD-1). Volume of distribution (approximately 2.5-3 l/kg) and the half-life (approximately 1 h) were similar. Urinary elimination of naltrexone was small, but also showed differences between the rats: HAD-1 > LAD-1 > Wistar, but with a statistically significant difference only between HAD-1 and Wistar rats. This study has therefore established the baseline disposition characteristics of naltrexone in these strains of rats.

pH-Sensitive Polymeric Nanoparticles Fabricated by Dispersion Polymerization for the Delivery of Bioactive Agents.

BACKGROUND: Development of pH-responsive nanoparticles capable of rapid degradation in the acidic environments in the endosomes and lysosomes of tumor tissues but relatively more stable in the physiological pH (pH 7.4) is desirable. OBJECTIVE: To show that the number of methoxy groups on the benzene ring of benzaldehyde bisacrylate acetal crosslinkers should affect the rate of hydrolysis of the crosslinkers and in vitro availability of the drug loaded into the nanoparticles. METHOD: Three pH-sensitive acetal crosslinkers were synthesized and characterized by 1H NMR, 13C NMR, FT-IR and high resolution mass spectroscopy (HR-MS). The nanoparticles were fabricated by free-radical dispersion polymerization method. Hydrolysis studies were carried out on the crosslinkers and nanoparticles; drug release studies were done on docetaxel-loaded nanoparticles at pH 5.0 and pH 7.4. The statisitical experimental design was randomized complete block design followed by analyses of variance with F-test of significance. Pairwise comparison test was used to locate specific differences among parameters of the crosslinkers and the nanopaticles. RESULTS: Scanning electron micrographs showed the formation of spherical particles. Particle size analysis showed that the nanoparticles are within nanosize range with negative zeta potential. Data showed that the rate of hydrolysis and drug release were faster at pH 5.0 compared to pH 7.4. Hydrolysis and drug release studies were dependent on the structure of the acetals: Di(2-methacryloyloxyethoxy)- [2,4,6-trimethoxyphenyl] methane crosslinker showed the fastest rate of hydrolysis, followed by di(2- methacryloyloxyethoxy)-[2,4-dimethoxyphe-nyl] methane and di(2-methacryloyloxyethoxy)-[4-methoxyphenyl] methane. CONCLUSION: The pH-responsive nanoparticles are suitable for the delivery of bioactive agents, especially anticancer drugs.

Antiretroviral Drugs-Loaded Nanoparticles Fabricated by Dispersion Polymerization with Potential for HIV/AIDS Treatment.

Highly active antiretroviral (ARV) therapy (HAART) for chronic suppression of HIV replication has revolutionized the treatment of HIV/AIDS. HAART is no panacea; treatments must be maintained for life. Although great progress has been made in ARV therapy, HIV continues to replicate in anatomical and intracellular sites where ARV drugs have restricted access. Nanotechnology has been considered a platform to circumvent some of the challenges in HIV/AIDS treatment. Dispersion polymerization was used to fabricate two types (PMM and ECA) of polymeric nanoparticles, and each was successfully loaded with four ARV drugs (zidovudine, lamivudine, nevirapine, and raltegravir), followed by physicochemical characterization: scanning electron microscope, particle size, zeta potential, drug loading, and in vitro availability. These nanoparticles efficiently inhibited HIV-1 infection in CEM T cells and peripheral blood mononuclear cells; they hold promise for the treatment of HIV/AIDS. The ARV-loaded nanoparticles with polyethylene glycol on the corona may facilitate tethering ligands for targeting specific receptors expressed on the cells of HIV reservoirs.

Computer Optimization of Biodegradable Nanoparticles Fabricated by Dispersion Polymerization.

Quality by design (QbD) in the pharmaceutical industry involves designing and developing drug formulations and manufacturing processes which ensure predefined drug product specifications. QbD helps to understand how process and formulation variables affect product characteristics and subsequent optimization of these variables vis-à-vis final specifications. Statistical design of experiments (DoE) identifies important parameters in a pharmaceutical dosage form design followed by optimizing the parameters with respect to certain specifications. DoE establishes in mathematical form the relationships between critical process parameters together with critical material attributes and critical quality attributes. We focused on the fabrication of biodegradable nanoparticles by dispersion polymerization. Aided by a statistical software, d-optimal mixture design was used to vary the components (crosslinker, initiator, stabilizer, and macromonomers) to obtain twenty nanoparticle formulations (PLLA-based nanoparticles) and thirty formulations (poly-ɛ-caprolactone-based nanoparticles). Scheffe polynomial models were generated to predict particle size (nm), zeta potential, and yield (%) as functions of the composition of the formulations. Simultaneous optimizations were carried out on the response variables. Solutions were returned from simultaneous optimization of the response variables for component combinations to (1) minimize nanoparticle size; (2) maximize the surface negative zeta potential; and (3) maximize percent yield to make the nanoparticle fabrication an economic proposition.

Design of naltrexone-loaded hydrolyzable crosslinked nanoparticles.

A hydrolyzable crosslinker (N,O-dimethacryloylhydroxylamine (MANHOMA)) was synthesized by a modified method and was characterized using 1H-NMR, FTIR, and melting point determination. Naltrexone-loaded nanoparticles were prepared by copolymerization of poly(ethylene glycol)1000 monomethyl ether mono methacrylate (PEO-MA), methyl methacrylate (MMA) and N,O-dimethacryloylhydroxylamine (MANHOMA) in 0.4% poly(vinyl alcohol) aqueous solution. The nanoparticles were characterized by FTIR, particle size determination and transmission electron microscope (TEM). The TEM photomicrographs of the nanoparticles show a crosslinked core surrounded by a ring formed by the polyethylene glycol tail of PEO-MA. The loading efficiency of the nanoparticles and in vitro drug availability from the nanoparticles were investigated. The naltrexone-loaded hydrolyzable crosslinked nanoparticles were able to sustain the release of naltrexone for different periods of time, depending on the monomer feed composition.

MS-Monitored Conjugation of Poly(ethylene glycol) Monomethacrylate to RGD Peptides.

Development of biologically active polymers is an active area of research due to their applications in varied and diverse fields of biomedical research: cell adhesion, tissue proliferation, and drug delivery. Recent advances in chemical modification allow fine-tuning of the properties of biomedical polymers to improve their applications: blood circulation half-life, stimuli-responsive degradation, site-specific targeting, drug loading, etc. In this article, convergent synthesis of polymerizable macromonomers bearing a site-specific ligand (RGD peptide) using a low molecular weight MA-poly(ethylene glycols) (PEGs) is presented. The method affords macromonomers useful as the starting materials to produce biomedical polymers. We found matrix assisted laser desorption/ionization mass spectromerty convenient in monitoring the conjugation process via step-by-step following of PEG modification.

Polylactide-based paclitaxel-loaded nanoparticles fabricated by dispersion polymerization: characterization, evaluation in cancer cell lines, and preliminary biodistribution studies.

The macromonomer method was used to prepare cross-linked, paclitaxel-loaded polylactide (PLA)-polyethylene glycol (stealth) nanoparticles using free-radical dispersion polymerization. The method can facilitate the attachment of other molecules to the nanoparticle surface to make it multifunctional. Proton nuclear magnetic resonance and Fourier transform infrared spectra confirm the synthesis of PLA macromonomer and cross-linking agent. The formation of stealth nanoparticles was confirmed by scanning and transmission electron microscopy. The drug release isotherm of paclitaxel-loaded nanoparticles shows that the encapsulated drug is released over 7 days. In vitro cytotoxicity assay in selected breast and ovarian cancer cell lines reveal that the blank nanoparticle is biocompatible compared with medium-only treated controls. In addition, the paclitaxel-loaded nanoparticles exhibit similar cytotoxicity compared with paclitaxel in solution. Confocal microscopy reveals that the nanoparticles are internalized by MCF-7 breast cancer cells within 1 h. Preliminary biodistribution studies also show nanoparticle accumulation in tumor xenograft model. The nanoparticles are suitable for the controlled delivery of bioactive agents.

Organic redox-initiated polymerization process for the fabrication of hydrogels for colon-specific drug delivery.

Organic-redox initiated polymerization technique based on the co-initiators system comprising benzoyl peroxide and N-phenyldiethanolamine was used at ambient temperature to fabricate pH-responsive hydrogels. The effects of changes in the concentration of the co-initiators system, the ratio in which the co-initiators combined, the type of the polymerization solvent, the pH of the hydrating medium, the concentration of the cross-linking agent based on azo-bond and the pH-sensitive cross-linking agent on the properties of the hydrogels were investigated. Increasing the concentration of the co-initiators system, decreasing the concentration of the two types of cross-linking agents, and replacing DMSO by ethanol as the polymerization solvent resulted in hydrogels with increased equilibrium swelling ratio and increased molecular weight between cross-links at pH 7.4. Increasing the concentration of N-phenyldiethanolamine while keeping the concentration of benzoyl peroxide constant gave hydrogels with increased equilibrium swelling ratios. The equilibrium swelling ratios of the hydrogels at pH 2.0 were not affected by the factors investigated. The polymerization technique may be suitable for the design of drug delivery systems containing thermolabile bioactive agents like peptides and proteins.