Combinatorial-Designed Epidermal Growth Factor Receptor-Targeted Chitosan Nanoparticles for Encapsulation and Delivery of Lipid-Modified Platinum Derivatives in Wild-Type and Resistant Non-Small-Cell Lung Cancer Cells

Development of efficient and versatile drug delivery platforms to overcome the physical and biological challenges in cancer therapeutics is an area of great interest, and novel materials are actively sought for such applications. Recent strides in polymer science have led to a combinatorial approach for generating a library of materials with different functional identities that can be “mixed and matched” to attain desired characteristics of a delivery vector. We have applied the combinatorial design to chitosan (CS), where the polymer backbone has been modified with polyethylene glycol, epidermal growth factor receptor-binding peptide, and lipid derivatives of varying chain length to encapsulate hydrophobic drugs. Cisplatin, <i>cis</i>-([PtCl₂(NH₃)₂]), is one of the most potent chemotherapy drugs broadly administered for cancer treatment. Cisplatin is a hydrophilic drug, and in order for it to be encapsulated in the developed nanosystems, it was modified with lipids of varying chain length. The library of four CS derivatives and six platinum derivatives was self-assembled in aqueous medium and evaluated for physicochemical characteristics and cytotoxic effects in platinum-sensitive and -resistant lung cancer cells. The results show that the lipid-modified platinate encapsulation into CS nanoparticles significantly improved cellular cytotoxicity of the drug. In this work, we have also reinforced the idea that CS is a multifaceted system that can be as successful in delivering small molecules as it has been as a nucleic acids carrier

<i>Mad2</i> Checkpoint Gene Silencing Using Epidermal Growth Factor Receptor-Targeted Chitosan Nanoparticles in Non-Small Cell Lung Cancer Model

RNA interference has emerged as a powerful strategy in cancer therapy because it allows silencing of specific genes associated with tumor progression and resistance. Mad2 is an essential mitotic checkpoint component required for accurate chromosome segregation during mitosis, and its complete abolition leads to cell death. We have developed an epidermal growth factor receptor (EGFR)-targeted chitosan system for silencing the <i>Mad2</i> gene as a strategy to efficiently induce cell death in EGFR overexpressing human A549 non-small cell lung cancer cells. Control and EGFR-targeted chitosan nanoparticles loaded with small interfering RNAs (siRNAs) against Mad2 were formulated and characterized for size, charge, morphology, and encapsulation efficiency. Qualitative and quantitative intracellular uptake studies by confocal imaging and flow cytometry, respectively, showed time-dependent enhanced and selective intracellular internalization of EGFR-targeted nanoparticles compared to nontargeted system. Targeted nanoparticles showed nearly complete depletion of Mad2 expression in A549 cells contrasting with the partial depletion in the nontargeted system. Accordingly, Mad2-silencing-induced apoptotic cell death was confirmed by cytotoxicity assay and flow cytometry. Our results demonstrate that EGFR-targeted chitosan loaded with <i>Mad2</i> siRNAs is a potent delivery system for selective killing of cancer cells

Stability Study Perspective of the Effect of Freeze-Drying Using Cryoprotectants on the Structure of Insulin Loaded into PLGA Nanoparticles

This work aimed to evaluate the influence of a freeze-drying process using different cryoprotectants on the structure of insulin loaded into poly­(lactic-<i>co</i>-glycolic acid) (PLGA) nanoparticles and to assess the stability of these nanoparticles upon 6 months of storage following ICH guidelines. Insulin-loaded PLGA nanoparticles with a size around 450 nm were dehydrated using a standard freeze-drying cycle, using trehalose, glucose, sucrose, fructose, and sorbitol at 10% (w/v) as cryoprotectants. All formulations, except those nonadded of cryoprotectant and added with trehalose, collapsed after freeze-drying. The addition of cryoprotectants increased the nanoparticles stability upon storage. FTIR results showed that insulin maintained its structure after encapsulation in about 88%, decreasing to 71% after freeze-drying. The addition of cryoprotectants prior to freeze-drying increased insulin structural stability an average of up to 79%. Formulations collapsed after freeze-drying showed better protein stabilization upon storage, in special sorbitol added formulation, preserving 76, 80, and 78% of insulin structure at 4 °C, 25 °C/60% RH, and 40 °C/75% RH, respectively. Principal component analysis also showed that the sorbitol-added formulation showed the most similar insulin structural modifications among the tested storage conditions. These findings suggested that regarding nanoparticles stability, cryoprotectants are versatile to be used in a standard freeze-drying, however they present different performances on the stabilization of the loaded protein. Thus, on the freeze-drying of the nanoparticles field, this work gives rise to the importance of the process of optimization, searching for a balance between a good obtainable cake with an optimal structural stabilization of the loaded protein

Supplementary material from Overcoming clofazimine intrinsic toxicity: statistical modelling and characterization of solid lipid nanoparticles

The aim of this work was to develop solid lipid nanoparticles (SLNs) loaded with clofazimine (CLZ) (SLNs-CLZ) to overcome its intrinsic toxicity and low water solubility, for oral drug delivery. A Box–Behnken design was constructed to unravel the relations between the independent variables in the selected responses. The optimized SLNs-CLZ exhibited the following properties: particle size <i>ca</i> 230 nm, zeta potential of −34.28 mV, association efficiency of 72% and drug loading of 2.4%, which are suitable for oral delivery. Further characterization included Fourier transformed infrared spectroscopy that confirmed the presence of the drug and the absence of chemical interactions. By differential scanning calorimetry was verified the amorphous state of CLZ. The storage stability studies ensured the stability of the systems over a period of 12 weeks at 4°C. <i>In vitro</i> cytotoxicity studies evidenced no effect of both drug-loaded and unloaded SLNs on MKN-28 gastric cells and on intestinal cells, namely Caco-2 and HT29-MTX cells up to 25 µg ml⁻¹ in CLZ. Free CLZ solutions exhibited IC₅₀ values of 16 and 20 µg ml⁻¹ for Caco-2 and HT29-MTX cells, respectively. It can be concluded that the optimized system, designed considering important variables for the formulation of poorly soluble drugs, represents a promising platform for oral CLZ delivery

Interactions of Microbicide Nanoparticles with a Simulated Vaginal Fluid

The interaction with cervicovaginal mucus presents the potential to impact the performance of drug nanocarriers. These systems must migrate through this biological fluid in order to deliver their drug payload to the underlying mucosal surface. We studied the ability of dapivirine-loaded polycaprolactone (PCL)-based nanoparticles (NPs) to interact with a simulated vaginal fluid (SVF) incorporating mucin. Different surface modifiers were used to produce NPs with either negative (poloxamer 338 NF and sodium lauryl sulfate) or positive (cetyltrimethylammonium bromide) surface charge. Studies were performed using the mucin particle method, rheological measurements, and real-time multiple particle tracking. Results showed that SVF presented rheological properties similar to those of human cervicovaginal mucus. Analysis of NP transport indicated mild interactions with mucin and low adhesive potential. In general, negatively charged NPs underwent subdiffusive transport in SVF, i.e., hindered as compared to their diffusion in water, but faster than for positively charged NPs. These differences were increased when the pH of SVF was changed from 4.2 to 7.0. Diffusivity was 50- and 172-fold lower in SVF at pH 4.2 than in water for negatively charged and positively charged NPs, respectively. At pH 7.0, this decrease was around 20- and 385-fold, respectively. The estimated times required to cross a layer of SVF were equal to or lower than 1.7 h for negatively charged NPs, while for positively charged NPs these values were equal to or higher than 7 h. Overall, our results suggest that negatively charged PCL NPs may be suitable to be used as carriers in order to deliver dapivirine and potentially other antiretroviral drugs to the cervicovaginal mucosal lining. Also, they further reinforce the importance in characterizing the interactions of nanosystems with mucus fluids or surrogates when considering mucosal drug delivery

<i>In Vitro</i> and <i>Ex Vivo</i> Evaluation of Polymeric Nanoparticles for Vaginal and Rectal Delivery of the Anti-HIV Drug Dapivirine

Prevention strategies such as the development of microbicides are thought to be valuable in the fight against HIV/AIDS. Despite recent achievements, there is still a long road ahead in the field, particularly at the level of drug formulation. Drug nanocarriers based on polymers may be useful in enhancing local drug delivery while limiting systemic exposure. We prepared differently surface-engineered poly­(ε-caprolactone) (PCL) nanoparticles (NPs) and tested their ability to modulate the permeability and retention of dapivirine in cell monolayers and pig vaginal and rectal mucosa. NPs coated with poly­(ethylene oxide) (PEO) were shown able to reduce permeability across monolayers/tissues, while modification of nanosystems with cetyl trimethylammonium bromide (CTAB) enhanced transport. In the case of coating NPs with sodium lauryl sulfate (SLS), dapivirine permeability was unchanged. All NPs increased monolayer/tissue drug retention as compared to unformulated dapivirine. This fact was associated, at least partially, to the ability of NPs to be taken up by cells or penetrate mucosal tissue. Cell and tissue toxicity was also affected differently by NPs: PEO modification decreased the <i>in vitro</i> (but not <i>ex vivo</i>) toxicity of dapivirine, while higher toxicity was generally observed for NPs coated with SLS or CTAB. Overall, presented results support that PCL nanoparticles are capable of modulating drug permeability and retention in cell monolayers and mucosal tissues relevant for vaginal and rectal delivery of microbicides. In particular, PEO-modified dapivirine-loaded PCL NPs may be advantageous in increasing drug residence at epithelial cell lines/mucosal tissues, which may potentially increase the efficacy of microbicide drugs

Pharmaceutical Formulations Containing Graphene and 5‑Fluorouracil for Light-Emitting Diode-Based Photochemotherapy of Skin Cancer

Nonmelanoma skin cancer (NMSC) is the most common cancer worldwide, among which 80% is basal cell carcinoma (BCC). Current therapies’ low efficacy, side effects, and high recurrence highlight the need for alternative treatments. In this work, a partially reduced nanographene oxide (p-rGOn) developed in our laboratory was used. It has been achieved through a controlled reduction of nanographene oxide via UV–C irradiation that yields small nanometric particles (below 200 nm) that preserve the original water stability while acquiring high light-to-heat conversion efficiency. The latter is explained by a loss of carbon–oxygen single bonds (C–O) and the re-establishment of sp2 carbon bonds. p-rGOn was incorporated into a Carbopol hydrogel together with the anticancer drug 5-fluorouracil (5-FU) to evaluate a possible combined PTT and chemotherapeutic effect. Carbopol/p-rGOn/5-FU hydrogels were considered noncytotoxic toward normal skin cells (HFF-1). However, when A-431 skin cancer cells were exposed to NIR irradiation for 30 min in the presence of Carbopol/p-rGOn/5-FU hydrogels, almost complete eradication was achieved after 72 h, with a 90% reduction in cell number and 80% cell death of the remaining cells after a single treatment. NIR irradiation was performed with a light-emitting diode (LED) system, developed in our laboratory, which allows adjustment of applied light doses to achieve a safe and selective treatment, instead of the standard laser systems that are associated with damages in the healthy tissues in the tumor surroundings. Those are the first graphene-based materials containing pharmaceutical formulations developed for BCC phototherapy

Microfluidic Assembly of a Multifunctional Tailorable Composite System Designed for Site Specific Combined Oral Delivery of Peptide Drugs

Multifunctional tailorable composite systems, specifically designed for oral dual-delivery of a peptide (glucagon-like peptide-1) and an enzymatic inhibitor (dipeptidyl peptidase 4 (DPP4)), were assembled through the microfluidics technique. Both drugs were coloaded into these systems for a synergistic therapeutic effect. The systems were composed of chitosan and cell-penetrating peptide modified poly(lactide-<i>co</i>-glycolide) and porous silicon nanoparticles as nanomatrices, further encapsulated in an enteric hydroxypropylmethylcellulose acetylsuccinate polymer. The developed multifunctional systems were pH-sensitive, inherited by the enteric polymer, enabling the release of the nanoparticles only in the simulated intestinal conditions. Moreover, the encapsulation into this polymer prevented the degradation of the nanoparticles’ modifications. These nanoparticles showed strong and higher interactions with the intestinal cells in comparison with the nonmodified ones. The presence of DPP4 inhibitor enhanced the peptide permeability across intestinal cell monolayers. Overall, this is a promising platform for simultaneously delivering two drugs from a single formulation. Through this approach peptides are expected to increase their bioavailability and efficiency <i>in vivo</i> both by their specific release at the intestinal level and also by the reduced enzymatic activity. The use of this platform, specifically in combination of the two antidiabetic drugs, has clinical potential for the therapy of type 2 diabetes mellitus