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Yufei Chen,1 Yannick Leandre Traore,1 Amanda Li,1 Keith R Fowke,2,3 Emmanuel A Ho1 1Laboratory for Drug Delivery and Biomaterials, Faculty of Pharmacy, 2Department of Medical Microbiology and Infectious Diseases, 3Department of Community Health Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada Abstract: Hydroxychloroquine (HCQ) has been shown to demonstrate anti-­inflammatory properties and direct anti-HIV activity. In this study, we describe for the first time the fabrication and in vitro evaluation of two types of intravaginal ring (IVR) devices (a surfaced-modified matrix IVR and a reservoir segmental IVR) for achieving sustained delivery (>14 days) of HCQ as a strategy for preventing male-to-female transmission of HIV. Both IVRs were fabricated by hot-melt injection molding. Surface-modified matrix IVRs with polyvinylpyrrolidone or poly(vinyl alcohol) coatings exhibited significantly reduced burst release on the first day (6.45% and 15.72% reduction, respectively). Reservoir IVR segments designed to release lower amounts of HCQ displayed near-zero-order release kinetics with an average release rate of 28.38 µg/mL per day for IVRs loaded with aqueous HCQ and 32.23 µg/mL per day for IVRs loaded with HCQ mixed with a rate-controlling excipient. Stability studies demonstrated that HCQ was stable in coated or noncoated IVRs for 30 days. The IVR segments had no significant effect on cell viability, pro-inflammatory cytokine production, or colony formation of vaginal and ectocervical epithelial cells. Both IVR systems may be suitable for the prevention of HIV transmission and other sexually transmitted infections. Keywords: intravaginal delivery, matrix system, reservoir system, polymeric drug carrier, drug release, microbicide, HIV/AIDS

Sidi Yang,1,2 Yufei Chen,1,2 Kaien Gu,1,2 Alicia Dash,1,2 Casey L Sayre,1 Neal M Davies,1 Emmanuel A Ho1,2 1Faculty of Pharmacy, 2Laboratory for Drug Delivery and Biomaterials, University of Manitoba, Winnipeg, MB, Canada Abstract: The goal of this study was to develop and characterize an intravaginal nanomedicine for the active targeted delivery of saquinavir (SQV) to CD4+ immune cells as a potential strategy to prevent or reduce HIV infection. The nanomedicine was formulated into a vaginal gel to provide ease in self-administration and to enhance retention within the vaginal tract. SQV-encapsulated nanoparticles (SQV-NPs) were prepared from poly(lactic-co-glycolic acid)(PLGA) and conjugated to antihuman anti-CD4 antibody. Antibody-conjugated SQV-NPs (Ab-SQV-NPs) had an encapsulation efficiency (EE%) of 74.4% ± 3.7% and an antibody conjugation efficiency (ACE%) of 80.95% ± 1.10%. Over 50% of total loaded SQV was released from NPs over 3 days. NPs were rapidly taken up by Sup-T1 cells, with more than a twofold increase in the intracellular levels of SQV when delivered by Ab-SQV-NPs in comparison to controls 1 hour post-treatment. No cytotoxicity was observed when vaginal epithelial cells were treated for 24 hours with drug-free Ab-NPs (1,000 µg/mL), 1% HEC placebo gel (200 mg/mL), or 1% HEC gel loaded with drug-free Ab-NPs (5 mg NPs/g gel, 200 mg/mL of gel mixture). Overall, we described an intravaginal nanomedicine that is nontoxic and can specifically deliver SQV into CD4+ immune cells. This platform may demonstrate potential utility in its application as postexposure prophylaxis for the treatment or reduction of HIV infection, but further studies are required. Keywords: nanoparticles, saquinavir, antibody conjugation, intravaginal gel, HIV/AIDS, microbicide

Conventional drugs have been facing various drug delivery obstacles, including first-pass metabolism for oral medications, drug degradation by cellular enzymes, off-target effects, and cytotoxicity of healthy cells. Nanoparticles (NP) application in drug delivery can compensate for these drawbacks to a great extent. NPs can be fabricated using different materials and structures to achieve desired therapeutic effects. For each type of NP material, its physicochemical properties determine compatibility with specific drugs and other supplemental compositions. The optimized material selection becomes prominent in NP development to improve NP performances. Due to the nature of NP fabrication, the process is long and expensive. To accelerate NP composition optimization, machine learning (ML) techniques are among the most promising methods for efficient data predictions and optimizations.As a proof-of concept, we created Gaussian Process (GP) models to make predictions for drug encapsulation efficiency (EE%) and therapeutic efficacy of 32 poly (lactic-co-glycolic acid) (PLGA) NPs that are formed with materials with different physicochemical properties. Two model drugs, doxorubicin (DOX) and docetaxel (DTX) were loaded separately. The IC50 values for the various NPs formulations were evaluated using the OVCAR3 epithelial ovarian cancer cell line. EE% GP model has the highest prediction accuracy with the lowest normalized root-mean-squared-error (RMSE) of 0.187. The DOX and DTX IC50 GP models have normalized RMSEs of 0.296 and 0.206, respectively, which are higher than that of the EE% GP model., Competing Interests: Declarations. Ethics approval: Not applicable. Consent to participate: Not applicable. Consent to publication: All the authors approve the publication. Competing interests: The authors declare no competing interests., (© 2024. Controlled Release Society.)

Ocular diseases have a major impact on patient's vision and quality of life, with approximately 2.2 billion people have visual impairment worldwide according to the findings from the World Health Organization (WHO). The eye is a complex organ with unique morphology and physiology consisting of numerous ocular barriers which hinders the entry of exogenous substances and impedes drug absorption. This in turn has a substantial impact on effective drug delivery to treat ocular diseases, especially intraocular disorders which has consistently presented a challenge to eye care professionals. The most common method of delivering medications to the eye is topical instillation of eye drops. Although this approach is a viable option for treating many ocular diseases remains a major challenge for the effective treatment of posterior ocular conditions. Up till now, incessant efforts have been committed to design innovative drug delivery systems with the hopes of potential clinical application. Modern developments in nanocarrier's technology present a potential chance to overcome these obstacles by enabling targeted delivery of the loaded medication to the eyes with improved solubility, delayed release, higher penetration and increased retention. This review covers the anatomy of eye with associated ocular barriers, ocular diseases and administration routes. In addition it primarily focuses on the latest progress and contemporary applications of ophthalmic formulations providing specific insight on nanostructured drug delivery carriers reported over the past 5 years highlighting their values in achieving efficient ocular drug delivery to both anterior and posterior segments. Most importantly, we outlined in this review the macro and nanotechnology based ophthalmic drug formulations that are being patented or marketed so far for treating ocular diseases. Finally, based on current trends and therapeutic concepts, we highlighted the challenges faced by novel ocular drug delivery systems and provided prospective future developments for further research in these directions. We hope that this review will serve as a source of motivation and ideas for formulation scientists in improving the design of innovative ophthalmic formulations., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: All authors approved the final manuscript. Competing interests: The authors declare no competing financial interests., (© 2024. Controlled Release Society.)

The COVID-19 outbreak has led to notable developments in point-of-care (POC) diagnostic devices, as they can be valuable resources in identifying and managing the spread of the pandemic. Currently, the majority of techniques demand advanced laboratory equipment and professionals to execute precise, efficient, accurate, and sensitive testing. In this work, we report a new method to significantly enhance the sensitivity of microwave sensing of the SARS-CoV-2 virus by functionalizing the sensor surface using anti-SARS-CoV-2 spike antibody-gold nanoparticle (AuNPs) conjugates. AuNPs were surface-functionalized with the antispike antibody by EDC/NHS chemistry via PEG as a linker to form the conjugate (Ab-PEG-AuNPs). The Ab-PEG-AuNPs nanoconjugate was then coated onto the sensor through APTES and used for selectively capturing the spike protein on the SARS-CoV-2 virus. The sensing performance of the modified sensor was demonstrated via both experimental measurements and numerical simulations. Our sensor exhibited high sensitivity, achieving a limit of detection of 1,000 copies/mL of the SARS-CoV-2 virus within a 60 min time frame while requiring a minimal sample volume of 100 μL. The sensor exhibits outstanding specificity in distinguishing SARS-CoV-2 from other viruses, including influenza A and B, SARS-CoV-1, and MERS-CoV. Overall, this sensor provides a sensitive and label-free alternative for COVID-19 POC diagnosis., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The bacterium Streptococcus pneumoniae has become a leading cause of meningitis, sepsis, and bacterial pneumonia worldwide, with increased prevalence of antibiotic-resistant serotypes serving to exacerbate the issue. The main factor responsible for colonization and immune response escape in pneumococcal infections is the secreted molecule pneumolysin, which is a subset within a family of related toxins that form transmembrane pores in biological membranes through cholesterol recognition and binding. The conserved activity and structure of pneumolysin between all observed S. pneumoniae serotypes, along with its requirement for pathogenicity, has made this molecule an attractive target for vaccination, diagnostic, and sequestration platforms, but not yet as a facilitative agent for therapeutic treatment. Consequently, the present work aimed to examine the impact of liposomal cholesterol content for pneumolysin-induced release of the encapsulated antimicrobial peptide nisin. It was determined that a cholesterol content above 45 mol% was necessary to facilitate interactions with both purified pneumolysin toxin and S. pneumoniae culture, demonstrated through enhanced nisin release and a reduction in hemolytic rates upon exposure of the toxin with cholesterol-rich vesicles. Antibacterial testing highlighted the ability of the developed platform to elicit a potent and specific bactericidal response in vitro against cultured S. pneumoniae when compared to a control strain, Staphylococcus epidermidis. It further improved viability of a fibroblast cell line upon S. pneumoniae challenge, outperforming free nisin via the synergistic impact of simultaneous bacterial clearance and pneumolysin neutralization. These findings collectively indicate that cholesterol-rich liposomes hold promise as a selective treatment platform against pneumococcal infections., (© 2024. Controlled Release Society.)

Bacterial vaginosis (BV) is a common vaginal infection affecting millions of women. Vaginal anaerobic dysbiosis occurs when Lactobacillus spp., the dominant flora in healthy vagina is replaced by certain overgrown anaerobes, resulting in unpleasant symptoms such as vaginal discharge and odor. With a high recurrence rate, BV also severely impacts the overall quality of life of childbearing women by inducing preterm delivery and increasing the risks of pelvic inflammatory disease and sexually transmitted infections. Among various BV-associated bacteria, Gardnerella vaginalis ( G. vaginalis ) has been identified as a primary pathogen since it has been isolated from almost all women carrying BV and exhibits higher virulence potential over other bacteria. When dealing with BV relapse, intravaginal drug delivery systems are superior to conventional oral antibiotic therapies in improving therapeutic efficacy owing to more effective drug dose, reduced drug resistance and minimized side effects such as stomach irritation. Traditional intravaginal drug administration generally involves solids, semi-solids and delivery devices inserted into the vaginal lumen to achieve sustained drug release. However, they are mostly designed for continuous drug release and are not preventative therapies, resulting in severe side effects caused by excess dosing. Stimuli-responsive systems that can release drug only when needed ('on-demand') can help diminish these negative side effects. Hence, we developed a bacteria-responsive liposomal platform for the prevention and treatment of BV. This platform demonstrated sustained drug release in the presence of vaginolysin, a toxin secreted specifically by G. vaginalis . We prepared four liposome formulations and evaluated their responsiveness to G. vaginalis . The results demonstrated that the liposome formulations could achieve cumulative drug release ranging from 46.7% to 51.8% over a 3-5 d period in response to G. vaginalis and hardly any drug release in the presence of Lactobacillus crispatus ( L. crispatus ), indicating the high specificity of the system. Overall, the bacteria-responsive drug release platform has great potential, since it will be the first time to realize sustained drug release stimulated by a specific pathogen for BV prevention and treatment. This on-demand therapy can potentially provide relief to the millions of women affected by BV., (Creative Commons Attribution license.)

Curcumin has been explored for its anti-cancer potential, but is severely limited by its hydrophobicity and sensitivity to light and water. In this study, poly (lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) were synthesized to encapsulate curcumin via single emulsion method to improve curcumin stability and bioavailability. The PLGA NPs were coated with oligomeric chitosan (COS) and RGD peptide (a peptide consisting of Arg-Gly-Asp) using amine-reactive chemistry (NHS and EDC). Both COS and RGD had been previously shown to accumulate and target many different types of cancer cells. NPs were characterised based on size distribution, zeta potential, and binding efficiency of RGD peptide. They were also evaluated on encapsulation efficiency, and stability, of curcumin within the NPs. OVCAR-3 cancer cells were treated with COS and RGD-coated PLGA NPs loaded with Coumarin-6 dye for fluorescent imaging of cell uptake. They were also treated with curcumin-loaded NPs to determine cytotoxicity and effectiveness of delivery. The NPs exhibited size distribution and zeta potential within expected values, though binding efficiency of RGD was low. Curcumin-loaded NPs showed significant increase in cytotoxicity over free (unencapsulated) curcumin, and void (empty) NPs, suggesting successful delivery of curcumin as an anti-cancer agent; the performance of COS and RGD coated NPs over bare PLGA NPs was inconclusive, however, optimization will be required to improve formulation during the coating steps. This method of NP synthesis serves as proof of concept for a modular solution to the development of various coated polymeric NPs for other drugs or applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wong and Ho.)

Additive manufacturing, commonly referred to as three-dimensional (3D) printing, has the potential to initiate a paradigm shift in the field of medicine and drug delivery. Ever since the advent of the first-ever United States Food and Drug Administration (US FDA)-approved 3D printed tablet, there has been an increased interest in the application of this technology in drug delivery and biomedical applications. 3D printing brings us one step closer to personalized medicine, hence rendering the "one size fits all" concept in drug dosing obsolete. In this review article, we focus on the recent developments in the field of modified drug delivery systems in which various types of additive manufacturing technologies are applied., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chan, Ranjitham Gopalakrishnan, Traore and Ho.)

Women are more susceptible to HIV transmission through unprotected heterosexual intercourse due to biological and social vulnerabilities. Intravaginal delivery of siRNAs targeting viral genes, host genes, or in combination has shown promising outcomes against HSV, HPV and HIV. Therefore, in this study, we designed, developed and evaluated a pH-sensitive RNAi-based combination nanomicrobide for the prevention/reduction of vaginal transmission of HIV. The nanomicrobide was composed of siRNA-PEI encapsulated PLGA-PEG nanoparticles (siRNA NP) loaded in a HEC gel dosage form with siRNA targeting host gene CCR5 and the viral gene Nef as a dual preventive strategy. Knocking down CCR5, a co-receptor for HIV could prevent HIV from attaching to and entering host cells and knocking down Nef could reactivate autophagy that was inhibited by Nef to improve the elimination of intracellular virus that escaped the first line of defense. The siRNA NP showed a desirable particle size and zeta potential for intravaginal delivery and a pH-dependent release profile whereby low amounts of siRNA was released under acidic vaginal conditions (vaginal fluid simulant; VFS, pH 4.2) (6.0 ± 0.4% released over 15 days) but significantly higher amounts of siRNA was released under neutral pH conditions (phosphate buffered saline; PBS, pH 7.4) (22.9 ± 0.4% released over 15 days). The CCR5-Nef-specific siRNA NP efficiently knocked down CCR5 and Nef protein expression by 43% and 63%, respectively, reactivated Nef-blocked autophagy and inhibited the replication of HIV in vitro (71.8% reduction in p24 expression). After being formulated into a gel dosage form, siRNA NP could be readily released from the gel, penetrate the vaginal epithelial layer, get taken up into the target cells and knockdown Nef and CCR5 without causing cytotoxicity in a vaginal mucosal co-culture model. Functionalization of siRNA NP with anti-CD4 antibody and loaded into a 0.5% HEC gel improved vaginal distribution and uptake of siRNA in a mouse model with distribution of siRNA restricted to the reproductive tract without any unwanted systemic uptake. The 0.5% HEC gel loaded with siRNA NP-(m)CD4 significantly downregulated approximately 40% of CCR5 protein in the lower vagina and 36% of CCR5 protein in the upper vaginal and cervical region. In contrast, 0.5% HEC gel loaded with siRNA NP-IgG did not result in significant gene knockdown., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Presently, different types of eye diseases, such as glaucoma, myopia, infection, and dry eyes are treated with topical eye drops. However, due to ocular surface barriers, eye drops require multiple administrations, which may cause several risks, thereby necessitating additional strategies. Some of the key characteristics of an ideal ocular drug delivery system are as follows: (a) good penetration into cornea, (b) high drug retention in the ocular tissues, (c) targetability to the desired regions of the eye, and (d) good bioavailability. It is worthy to note that the corneal epithelial tight junctions hinder the permeation of therapeutics through the cornea. Therefore, it is necessary to design nanocarriers that can overcome these barriers and enhance drug penetration into the inner parts of the eye. Moreover, intelligent multifunctional nanocarriers can be designed to include cavities, which may help encapsulate sufficient amount of the drug. In addition, nanocarriers can be modified with the targeting moieties. Different types of nanocarriers have been developed for ocular drug delivery applications, including emulsions, liposomes, micelles, and nanoparticles. However, these formulations may be rapidly cleared from the eye. The therapeutic use of the nanoparticles (NPs) is also hindered by the non-specific adsorption of proteins on NPs, which may limit their interaction with the cellular moieties or other targeted biological factors. Functional drug delivery systems (DDS), which can offer targeted ocular drug delivery while avoiding the non-specific protein adsorption could exhibit great potential. This could be further realized by the on-demand DDS, which can respond to the stimuli in a spatio-temporal fashion. The cell-mediated DDS offer another valuable platform for ophthalmological drug delivery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in an unprecedented economic and societal impact. Developing simple and accurate testing methods for point-of-care (POC) diagnosis is crucial not only for the control of COVID-19, but also for better response to similar outbreaks in the future. In this work, we present a novel proof-of-concept of a microfluidic microwave sensing method for POC diagnosis of the SARS-CoV-2 virus. This method relies on the antibody immobilized on the microwave sensor to selectively capture and concentrate the SARS-CoV-2 antigen or virus present in a buffer solution flowing through the sensor region in a microchannel. The capturing of the SARS-CoV-2 antigen or virus results in a change in the permittivity of the medium near the sensor region reflected by the resonance frequency shift which is used for detection. The use of microchannels offers precise control of the sample volume and the continuous flow nature also offers the potential to monitor the dynamic capturing process. The microwave-microfluidic device shows a good sensitivity of 0.1 ng ml -1 for the SARS-CoV-2 antigen and 4000 copies per ml for the SARS-CoV-2 virus. The resonance frequency shift presents a linear relationship with the logarithm of antigen or virus concentration, respectively. This detection method is able to distinguish SARS-CoV-2 from the antigen of human CD4 and two human coronaviruses (MERS and HKU1), which presents a new pathway towards POC diagnosis of the COVID-19 at the community level. It presents the potential to detect other viruses by functionalizing the microwave sensor with respective antibodies.

Vaginal drug delivery has been shown to be a promising strategy for the prevention of sexually transmitted infections. Therapy delivered at the site of infection has many advantages including improved therapeutic efficacy, reduction in systemic toxicity, and reduced potential for development of drug resistance. We developed a "smart" combination intravaginal ring (IVR) that will (1) provide continuous release of hydroxychloroquine (HCQ) to induce T cell immune quiescence as the first-line of defense and (2) release nanoparticles containing anti-CCR5 siRNA only during sexual intercourse when triggered by the presence of seminal fluid as the second-line of defense. The IVR was capable of releasing HCQ over 25 days with a mean daily release of 31.17 ± 3.06 µg/mL. In the presence of vaginal fluid simulant plus seminal fluid simulant, over 12 × more nanoparticles (5.12 ± 0.9 mg) were released over a 4-h period in comparison to IVR segments that were incubated in the presence of vaginal fluid simulant alone (0.42 ± 0.19 mg). Anti-CCR5 siRNA nanoparticles were able to knockdown 83 ± 5.1% of CCR5 gene expression in vitro in the CD4 + T cell line Sup-T1. The IVR system also demonstrated to be non-cytotoxic to VK2/E6E7 vaginal epithelial cells., (© 2021. The Author(s).)

We designed and engineered novel intravaginal ring (IVR) medical devices via fused deposition modeling (FDM) three-dimensional (3D) printing for controlled delivery of hydroxychloroquine, IgG, gp120 fragment (encompassing the CD4 binding site), and coumarin 6 PLGA-PEG nanoparticles (C6NP). The hydrophilic polyurethanes were utilized to 3D-print reservoir-type IVRs containing a tunable release controlling membrane (RCM) with varying thickness and adaptable micro porous structures (by altering the printing patterns and interior fill densities) for controlled sustained drug delivery over 14 days. FDM 3D printing of IVRs were optimized and implemented using a lab-developed Cartesian 3D printer. The structures were investigated by scanning electron microscopy (SEM) imaging and in vitro release was performed using 5 mL of daily-replenished vaginal fluid simulant (pH 4.2). The release kinetics of the IVR segments were tunable with various RCM (outer diameter to inner diameter ratio ranging from 1.12 to 2.61) produced from FDM 3D printing by controlling the printing perimeter to provide daily zero-order release of HCQ ranging from 23.54 ± 3.54 to 261.09 ± 32.49 µg/mL/day. IgG, gp120 fragment, and C6NP release rates demonstrated pattern and in-fill density-dependent characteristics. The current study demonstrated the utility of FDM 3D printing to rapidly fabricate complex micro-structures for tunable and sustained delivery of a variety of compounds including HCQ, IgG, gp120 fragment, and C6NP from IVRs in a controlled manner., (© 2022. The Author(s).)

Doxorubicin (DOX) is a natural antibiotic with antineoplastic activity. It has been used for over 40 years and remains one of the most used drugs in chemotherapy for a variety of cancers. However, cardiotoxicity limits its use for long periods. To overcome this limitation, encapsulation in smart drug delivery systems (DDS) brings advantages in comparison with free drug administration (i.e., conventional anticancer drug therapy). In this review, we present the most relevant nanostructures used for DOX encapsulation over the last 10 years, such as liposomes, micelles and polymeric vesicles (i.e., polymersomes), micro/nanoemulsions, different types of polymeric nanoparticles and hydrogel nanoparticles, as well as novel approaches for DOX encapsulation. The studies highlighted here show these nanoformulations achieved higher solubility, improved tumor cytotoxicity, prolonged DOX release, as well as reduced side effects, among other interesting advantages., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study develops mechanistic understanding of the factors which control the phase in syntheses of copper selenide nanocrystals by investigating how the chemistry of the dodecylselenol reactant is altered by the ligand and solvent environment. 1 H NMR and 77 Se NMR were used to study how commonly used solvents (octadecene and dioctylether) and ligands (oleylamine, oleic acid, stearylamine, stearic acid and trioctyl phosphine) change the nature of the dodecylselenol reactant at 25 °C, 155 °C and 220 °C. Unsaturations were prone to selenol additons, carboxylates underwent selenoesterification, amines caused the release of H 2 Se gas, and the phosphine formed phosphine selenide. Adventitious water caused oxidation to didodecyldiselenide. NMR studies were correlated with the phases that resulted in syntheses of nanocrystalline copper selenides, in which berzalianite, umangite or a metastable hexagonal phase were produced as identified by X-ray diffraction, depending on the ligand and solvent environemnts. Formation of the rare hexagonal Cu 2- x Se phase could be assigned to cases that included DD 2 Se 2 as a reactive intermediate, or strong L-type ligation of amines which was dependant on alkyl chain length.

Introduction: Acetylsalicylic acid (ASA) is a well-known and safe anti-inflammatory. At low-dose, it is prescribed to prevent secondary cardiovascular events in those with pre-existing conditions and to prevent preeclampsia. Little is known about how low-dose ASA affects the immune response. In this study, we followed women to assess how ASA use modifies T cells immune phenotypes in the blood and at the genital tract., Methods: HIV uninfected women from Kenya were enrolled in this study and followed for one month to assess baseline responses including systemic/mucosal baseline immune activation. Participants then received 81mg of ASA daily for 6 weeks to assess changes to T cell immune activation (systemic and mucosal) relative to baseline levels., Results: The concentration of ASA measured in the blood was 58% higher than the level measured at the female genital tract. In the blood, the level of ASA was inversely correlated with the following: the proportion of Th17 expressing HLA-DR (p=0.04), the proportion of effector CD4 + T cells expressing CCR5 (p=0.03) and the proportion of CD8 + Tc17 expressing CCR5 (p=0.04). At the genital tract, ASA use correlated with a decreased of activated CD4 + T cells [CD4 + CCR5 + CD161 + (p=0.02) and CD4 + CCR5 + CD95 + (p=0.001)]., Conclusion: This study shows that ASA use impacts the immune response in both the systemic and genital tract compartments. This could have major implications for the prevention of infectious diseases such as HIV, in which the virus targets activated T cells to establish an infection. This could inform guidelines on ASA use in women., Clinical Trial Registration: ClinicalTrials.gov, identifier NCT02079077., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lajoie, Kowatsch, Mwangi, Boily-Larouche, Oyugi, Chen, Kimani, Ho, Kimani and Fowke.)

A review on microfluidic technology for antibacterial resistance study and antibiotic susceptibility testing (AST) is presented here. Antibiotic resistance has become a global health crisis in recent decades, severely threatening public health, patient care, economic growth, and even national security. It is extremely urgent that antibiotic resistance be well looked into and aggressively combated in order for us to survive this crisis. AST has been routinely utilized in determining bacterial susceptibility to antibiotics and identifying potential resistance. Yet conventional methods for AST are increasingly incompetent due to unsatisfactory test speed, high cost, and deficient reliability. Microfluidics has emerged as a powerful and very promising platform technology that has proven capable of addressing the limitation of conventional methods and advancing AST to a new level. Besides, potential technical challenges that are likely to hinder the development of microfluidic technology aimed at AST are observed and discussed. To conclude, it is noted that (1) the translation of microfluidic innovations from laboratories to be ready AST platforms remains a lengthy journey and (2) ensuring all relevant parties engaged in a collaborative and unified mode is foundational to the successful incubation of commercial microfluidic platforms for AST.

Exosomes are extracellular vesicles released from cells and are characterized by a lipid bilayer membrane encapsulating a variety of biological molecules such as nucleic acids or proteins within the lumen or the lipid-bilayer. Under physiological environments, exosomes mediate cell-to-cell communication and cargo transport. Therefore, exosomes have been explored as drug delivery vehicles for improving therapeutic outcomes. Although recent studies have demonstrated promising advances with exosome-based drug delivery systems, several challenges severely hinder further development of exosomes for clinical applications. This review summarizes and emphasizes some of the technical challenges related to the isolation, characterization, and stability testing of exosomes. More importantly, challenges related specifically to the application of exosomes for drug delivery such as cell-uptake, drug loading, drug release, and in vivo distribution will be examined in this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Intravenous administration of anti-α 4 β 7 monoclonal antibody in macaques decreases simian immunodeficiency virus (SIV) vaginal infection and reduces gut SIV loads. Because of potential side effects of systemic administration, a prophylactic strategy based on mucosal administration of anti-α 4 β 7 antibody may be safer and more effective. With this in mind, we developed a novel intravaginal formulation consisting of anti-α 4 β 7 monoclonal antibody-conjugated nanoparticles (NPs) loaded in a 1% hydroxyethylcellulose (HEC) gel (NP-α 4 β 7 gel). When intravaginally administered as a single dose in a rhesus macaque model, the formulation preferentially bound to CD4 + or CD3 + T cells expressing high levels of α 4 β 7 , and occupied ~40% of α 4 β 7 expressed by these subsets and ~25% of all cells expressing α 4 β 7 Blocking of the α 4 β 7 was restricted to the vaginal tract without any changes detected systemically., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

C. trachomatis is the most common sexually transmitted bacterial infection in the world. Although the infection can be easily controlled by the use of antibiotics, several reports of clinical isolates that are resistant to antibiotics have prompted us to search for alternative strategies to manage this disease. In this paper, we developed a nanoparticle formulation (PDGFR-β siRNA-PEI-PLGA-PEG NP) that can simultaneously induce autophagy in human cells and knock down PDGFR-β gene expression, an important surface binding protein for C. trachomatis, as a strategy to reduce vaginal infection of C. trachomatis. PDGFR-β siRNA-PEI-PLGA-PEG NP significantly induced autophagy in human vaginal epithelial cells (VK2/E6E7) 48 hr post treatment by improving autophagic degradation activity without causing inflammation, apoptosis or any decrease in cell viability. Beclin-1, VPS34 (markers for initiation stage of autophagy), UVRAG, TECPR-1 (markers for degradation stage of autophagy) were found to be significantly upregulated after treatment with PDGFR-β siRNA-PEI-PLGA-PEG NP. Furthermore, PDGFR-β siRNA-PEI-PLGA-PEG NP decreased PDGFR-β mRNA expression by 50% and protein expression by 43% in VK2/E6E7 cells 48 hr post treatment. Treatment of cells with PDGFR-β siRNA-PEI-PLGA-PEG NP significantly decreased the intracellular C. trachomatis and extracellular release of C. trachomatis by approximately 65% and 67%, respectively, in vitro through augmenting autophagic degradation pathways and reducing bacterial binding simultaneously.

The objective of this study was to develop and characterize a novel intravaginal membrane platform for pH-triggered release of nanoparticles (NPs), which is essential for efficient intravaginal delivery of certain effective but acid-labile therapeutic agents for sexually transmitted infections, such as small interfering RNA (siRNA). A pH-responsive polyurethane (PU) was electrospun into a porous nanofibrous membrane. The diameters of the fibers, as well as the thickness and pore sizes of the membrane under dry and wet conditions (pH 4.5 and 7.0), were determined from scanning electron microscopy (SEM) micrographs. pH-dependent zeta-potential (ζ) of the membrane was evaluated using a SurPASS electrokinetic analyzer. Visiblex™ color-dyed polystyrene NPs (PSNs, 200 nm, COOH) and CCR5 siRNA-encapsulated solid lipid NPs (SLNs) were used for in vitro NP release studies in a vaginal fluid simulant (VFS) at pH 4.5 (normal physiological vaginal pH) and 7.0 (vaginal pH neutralization by semen). During 24 h of incubation in VFS, close-to-zero PSNs (2 ± 1%) and 28 ± 4% SLNs were released through the PU membrane at pH 4.5, whereas the release of PSNs and SLNs significantly increased to 60 ± 6% and 59 ± 8% at pH 7.0, respectively. The pH-responsive release of NPs hinged on the electrostatic interaction between the pH-responsive membrane and the anionic NPs, and the change in pH-responsive morphology of the membrane. In vitro biocompatibility studies of the membrane showed no significant cytotoxicity to VK2/E6E7 human epithelial cells and Sup-T1 human T-cells and no significant changes in the expression of pro-inflammatory cytokines (IL-6, IL-8, and IL-1β). Overall, the porous pH-responsive PU membrane demonstrated its potential in serving as a "window" membrane in reservoir-type intravaginal rings (IVRs) for pH-responsive intravaginal release of NPs. STATEMENT OF SIGNIFICANCE: Stimuli-responsive intravaginal nanoparticle release is achieved for the first time through a new electrospun pH-responsive polyurethane (PU) semi-permeable membrane, which can serve as a "window" membrane in the reservoir-type IVR for the prevention of human immunodeficiency virus (HIV) transmission. Almost no release of nanoparticles was observed at normal pH in the female genital tract (in vaginal fluid simulant [VFS], at pH 4.5); however, a continuous release of NPs was observed at elevated pH in the female genital tract (in VFS, at pH 7.0). This pH-responsive intravaginal release can reduce side effect and drug resistance by avoiding unnecessary exposure. The PU semi-permeable membrane demonstrated potential use as biomaterials for "smart" intravaginal nanoparticle release and has great potential to protect women from HIV., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Efforts in developing an effective vaccine for human immunodeficiency virus (HIV) has been challenging as HIV strains are highly variable and exhibit extraordinary mutability. Despite condom usage and pre-exposure prophylaxis as excellent prevention strategies, lack of accessibility in some developing countries and low adherence due to sociocultural factors continue to act as barriers in reducing the HIV epidemic. Microbicides are topical therapies developed to prevent HIV and other sexually transmitted infections during intercourse. Microbicides applied vaginally or rectally are intended to prevent HIV infection at the site of transmission by either inhibiting its entry into immune cells or prevent viral replication. This review will summarize some of the current state-of-the-art microbicide formulations that are in preclinical and clinical stages of development and discuss some of the challenges associated with microbicide development., (© 2018 American Society for Clinical Pharmacology and Therapeutics.)

To achieve a pH-responsive switchable on-demand release of nanoparticles (NPs) from intravaginal rings (IVR), a new pH-sensitive polyurethane (PU) bearing dimethylolpropionic acid (PEG-DMPA-HDI-PG) was synthesized to encapsulate NPs as a physically cross-linked hydrogel within a segmented reservoir-IVR. A new PEGylated polyaspartic acid-based copolymer conjugated with the fluorescent dye Orange II (PASP-PEG-Ph-Orange) was synthesized to self-assemble in aqueous solution into NPs (251-283 nm) for the release study. Chemical structures of the PEG-DMPA-HDI-PG and PASP-PEG-Ph-Orange were confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) and 1 H nuclear magnetic resonance ( 1 H NMR) spectroscopy. PASP-PEG-Ph-Orange NPs showed the highest fluorescent emission at 570 nm for tracking, and PEG-DMPA-HDI-PG became a pH-responsive supramolecular hydrogel in distilled water at 20 wt %. PASP-PEG-Ph-Orange NPs were blended with the PEG-DMPA-HDI-PG hydrogel to form an inclusion complex and then filled into segmented reservoir-IVRs containing two 1/32 in. diameter holes. The segmented IVR filled with the NP encapsulated hydrogel showed continuous release of the NPs at pH 7.0 but a close-to-zero release at pH 4.2 for 12 h and, moreover, demonstrated a pH-responsive switchable on-demand NPs release. The PASP-PEG-Ph-Orange and PEG-DMPA-HDI-PG showed no and low cytotoxicity toward the human vaginal epithelial cell line VK2/E6E7, respectively. Overall, the segmented IVR filled with PEG-DMPA-HDI-PG hydrogel demonstrated its potential use for the switchable on-demand intravaginal release of nanocarriers.

Introduction: At its basic level, HIV infection requires a replication-competent virus and a susceptible target cell. Elevated levels of vaginal inflammation has been associated with the increased risk of HIV infection as it brings highly activated HIV target cells (CCR5+CD4+ T cells; CCR5+CD4+CD161+ Th17 T cells) to the female genital tract (FGT) where they interact with HIV. Decreased HIV risk has been associated with a phenotype of decreased immune activation, called immune quiescence, described among Kenyan female sex workers who were intensely exposed to HIV yet remain uninfected. Current prevention approaches focus on limiting viral access. We took the novel HIV prevention approach of trying to limit the number of HIV target cells in the genital tract by reducing inflammation using safe, affordable and globally accessible anti-inflammatory drugs., Methods: We hypothesized that the daily administration of low doses of acetylsalicylic acid (ASA 81 mg) or hydroxychloroquine (HCQ 200 mg) would reduce inflammation thereby decreasing HIV target cells at the FGT. Low-risk HIV seronegative women from Nairobi, Kenya were randomized for six weeks therapy of ASA (n = 37) or HCQ (n = 39) and tested to determine the impact on their systemic and mucosal immune environment., Results: The results showed that HCQ use was associated with a significant reduction in the proportion of systemic T cells that were CCR5+CD4+ (p = 0.01) and Th17 (p = 0.01). In the ASA arm, there was a 35% and 28% decrease in the proportion of genital T cells that were CD4+CCR5+ (p = 0.017) and Th17 (p = 0.04) respectively. Proteomic analyses of the cervical lavage showed ASA use was associated with significantly reduced amount of proteins involved in the inflammatory response and cell recruitment at the mucosa, although none of the individual proteins passed multiple comparison correction. These changes were more apparent in women with Lactobacillus dominant microbiomes., Conclusion: Together, these data indicate that taking low-dose ASA daily was associated with significant reduction in HIV target cells at the FGT. This study provides proof-of-concept for a novel HIV-prevention approach that reducing inflammation using safe, affordable and globally accessible non-steroidal anti-inflammatory agents is associated with significant reduction in the proportion of HIV-target cells at the FGT., (© 2018 The Authors. Journal of the International AIDS Society published by John Wiley & sons Ltd on behalf of the International AIDS Society.)

Evidence suggests that women who are naturally resistant to HIV infection exhibit low baseline immune activation at the female genital tract (FGT). This "immune quiescent" state is associated with lower expression of T-cell activation markers, reduced levels of gene transcription and pro-inflammatory cytokine or chemokine production involved in HIV infection while maintaining an intact immune response against pathogens. Therefore, if this unique immune quiescent state can be pharmacologically induced locally, it will provide an excellent women-oriented strategy against HIV infection To our knowledge, this is the first research article evaluating in vivo, an innovative trackable implant that can provide controlled delivery of hydroxychloroquine (HCQ) to successfully attenuate vaginal T lymphocyte activation and inflammation in a rabbit model as a potential strategy to induce an "immune quiescent" state within the FGT for the prevention of HIV infection. This biocompatible implant can deliver HCQ above therapeutic concentrations in a controlled manner, reduce submucosal immune cell recruitment, improve mucosal epithelium integrity, decrease protein and gene expression of T-cell activation markers, and attenuate the induction of key pro-inflammatory mediators. Our results suggest that microbicides designed to maintain a low level of immune activation at the FGT may offer a promising new strategy for reducing HIV infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, we investigated the viscoelastic and mechanical behaviour of polyvinyl alcohol films formulated along with carrageenan, plasticizing agents (polyethylene glycol and glycerol), and when loaded with nanoparticles as a model for potential applications as microbicides. The storage modulus, loss modulus and glass transition temperature were determined using a dynamic mechanical analyzer. Films fabricated from 2% to 5% polyvinyl alcohol containing 3 mg or 5 mg of fluorescently labeled nanoparticles were evaluated. The storage modulus and loss modulus values of blank films were shown to be higher than the nanoparticle-loaded films. Glass transition temperature determined using the storage modulus, and loss modulus was between 40-50℃ and 35-40℃, respectively. The tensile properties evaluated showed that 2% polyvinyl alcohol films were more elastic but less resistant to breaking compared to 5% polyvinyl alcohol films (2% films break around 1 N load and 5% films break around 7 N load). To our knowledge, this is the first study to evaluate the influence of nanoparticle and film composition on the physico-mechanical properties of polymeric films for vaginal drug delivery.

Antibiotic alternatives are in great need for combating antibiotic resistance. Selective delivery of a potent non-selective non-resistance-inducing biocide (C17) to MRSA was achieved by encapsulating it in solid lipid nanoparticles (SLNs) conjugated with a MRSA-specific antibody (termed as "Ab"). The C17-loaded Ab-conjugated SLNs (C17-SLN-Ab) demonstrated significantly better antimicrobial activity than its antibody free counterpart (C17-loaded SLN) and C17-loaded SLN with a non-specific IgG antibody. In a new MRSA/fibroblast co-culture assay, C17-SLN-Ab showed selective toxicity toward MRSA than fibroblast cells. C17-SLN-Ab possesses double selectivity, exhibiting higher toxicity to MRSA than to Pseudomonas aeruginosa. This same strategy was used to successfully increase C17's selectivity against E. coli K12 by switching the conjugated anti-MRSA antibody to an anti-E. coli antibody, demonstrating versatility of this new strategy. This proof-of-concept research can be extended to other non-selective antimicrobials, against which bacterial resistance is unlikely to develop, to generate a new group of promising antibiotic alternatives., (Copyright © 2017 Elsevier Inc. All rights reserved.)

New amphiphilic PEGylated poly(aspartic acid) graft copolymer (PASP-PEG-Ph) was synthesized as a nanocarrier for intravaginal drug delivery of poorly water-soluble drugs. PASP-PEG-Ph self-assembled into negatively charged spherically shaped nanoparticles in the presence of pH 4.5 and pH 7.0 vaginal fluid simulants with a diameter of approximately 200 nm as evidenced by Zeta-potentiometer, scanning electron microscope (SEM), dynamic light scattering (DLS) analysis. A significant number of stable NPs could be maintained at pH 4.5, 37 °C for 13 days. The PASP-PEG-Ph NP showed no significant cytotoxicity toward the T-cell line SupT1 and human vaginal epithelial cell line Vk2/E6E7 up to 1 mg/mL. The highest encapsulation efficiency of the model drug coumarin 6 (C6) by PASP-PEG-Ph was 92.0 ± 5.7%. The sustained release profile of the encapsulated C6 was demonstrated by an in vitro release study. An in vitro cellular uptake study revealed strong cellular uptake of the C6 loaded NP by SupT1 cells within 2 h.

Tetrahydrocurcumin (THC), curcumin and calebin-A are curcuminoids found in turmeric ( Curcuma longa ). Curcuminoids have been established to have a variety of pharmacological activities and are used as natural health supplements. The purpose of this study was to identify the metabolism, excretion, antioxidant, anti-inflammatory and anticancer properties of these curcuminoids and to determine disposition of THC in rats after oral administration. We developed a UHPLC-MS/MS assay for THC in rat serum and urine. THC shows multiple redistribution phases with corresponding increases in urinary excretion rate. In-vitro antioxidant activity, histone deacetylase (HDAC) activity, histone acetyltransferase (HAT) activity and anti-inflammatory inhibitory activity were examined using commercial assay kits. Anticancer activity was determined in Sup-T1 lymphoma cells. Our results indicate THC was poorly absorbed after oral administration and primarily excreted via non-renal routes. All curcuminoids exhibited multiple pharmacological effects in vitro, including potent antioxidant activity as well as inhibition of CYP2C9, CYP3A4 and lipoxygenase activity without affecting the release of TNF-α. Unlike curcumin and calebin-A, THC did not inhibit HDAC1 and PCAF and displayed a weaker growth inhibition activity against Sup-T1 cells. We show evidence for the first time that curcumin and calebin-A inhibit HAT and PCAF, possibly through a Michael-addition mechanism., Competing Interests: This work was partially funded by the Sabinsa Corporation to Neal M. Davies and the authors Kalyanam Nagabushnam and Muhammed Majeed are employed with this company that supplied the tetrahydrocurcumin, curcumin and calebin-A materials for this study. All other authors declare no conflicts of interest. The authors alone are responsible for the content, writing, and interpretations in this paper.

RNA interference (RNAi)-mediated gene silencing offers a novel treatment and prevention strategy for human immunodeficiency virus (HIV) infection. HIV was found to infect and replicate in human brain cells and can cause neuroinfections and neurological deterioration. We designed dual-antibody-modified chitosan/small interfering RNA (siRNA) nanoparticles to deliver siRNA across the blood-brain barrier (BBB) targeting HIV-infected brain astrocytes as a strategy for inhibiting HIV replication. We hypothesized that transferrin antibody and bradykinin B2 antibody could specifically bind to the transferrin receptor (TfR) and bradykinin B2 receptor (B2R), respectively, and deliver siRNA across the BBB into astrocytes as potential targeting ligands. In this study, chitosan nanoparticles (CS-NPs) were prepared by a complex coacervation method in the presence of siRNA, and antibody was chemically conjugated to the nanoparticles. The antibody-modified chitosan nanoparticles (Ab-CS-NPs) were spherical in shape, with an average particle size of 235.7 ± 10.2 nm and a zeta potential of 22.88 ± 1.78 mV. The therapeutic potential of the nanoparticles was evaluated based on their cellular uptake and gene silencing efficiency. Cellular accumulation and gene silencing efficiency of Ab-CS-NPs in astrocytes were significantly improved compared to non-modified CS-NPs and single-antibody-modified CS-NPs. These results suggest that the combination of anti-Tf antibody and anti-B2 antibody significantly increased the knockdown effect of siRNA-loaded nanoparticles. Thus, antibody-mediated dual-targeting nanoparticles are an efficient and promising delivery strategy for inhibiting HIV replication in astrocytes. Graphical abstract Graphic representation of dual-antibody-conjugated chitosan nanoparticles for the targeted delivery of siRNA across the blood-brain barrier (BBB) for inhibiting HIV replication in astrocytes. a Nanoparticle delivery to the BBB and penetration. b TfR-mediated transcytosis of nanoparticles across the epithelial cells. c B2R-mediated endocytosis of nanoparticles in astrocytes. d The molecular interactions between HIV-1 Tat protein and Cyclin T1 and Tip110 cellular proteins. e A schematic representation of chitosan nanoparticles with its components. RNAPII RNA polymerase II, TAR transactivation response RNA element, LTR long terminal repeat, Ab antibody, CS chitosan, TPP tripolyphosphate.

To provide better protection for women against sexually transmitted infections, on-demand intravaginal drug delivery was attempted by synthesizing reversibly pH-sensitive polyether-polyurethane copolymers using poly(ethylene glycol) (PEG) and 1,4-bis(2-hydroxyethyl)piperazine (HEP). Chemical structure and thermo-characteristics of the synthesized polyurethanes were confirmed by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), 1 H-nuclear magnetic resonance ( 1 H-NMR), and melting point testing. Membranes were cast by solvent evaporation method using the prepared pH-sensitive polyurethanes. The impact of varying pH on membrane swelling and surface morphology was evaluated via swelling ratio change and scanning electron microscopy (SEM). The prepared pH-responsive membranes showed two times higher swelling ratio at pH 4 than pH 7 and pH-triggered switchable surface morphology change. The anionic anti-inflammatory drug diclofenac sodium (NaDF) was used as a model compound for release studies. The prepared pH-responsive polyurethane membranes allowed continuous NaDF release for 24h and around 20% release of total NaDF within 3h at pH 7 but little-to-no drug release at pH 4.5. NaDF permeation across the prepared membranes demonstrated a reversible pH-responsiveness. The pH-responsive polyurethane membranes did not show any noticeable negative impact on vaginal epithelial cell viability or induction of pro-inflammatory cytokine production compared to controls. Overall, the non-cytotoxic HEP-based pH-responsive polyurethane demonstrated its potential to be used in membrane-based implants such as intravaginal rings to achieve on-demand "on-and-off" intravaginal drug delivery., Statement of Significance: A reversible and sharp switch between "off" and "on" drug release is achieved for the first time through new pH-sensitive polyurethane membranes, which can serve as window membranes in reservoir-type intravaginal rings for on-demand drug delivery to prevent sexually transmitted infections (STIs). Close to zero drug release occurs at the normal vaginal pH (4.5) for minimal side effects. Drug release is only triggered by elevation of pH to 7 during heterosexual intercourse. The reversibly sharp and fast "on-and-off" switch arises from the creative incorporation of a pH-sensitive monomer in the soft segment of polyurethane. This polyurethane biomaterial holds great potential to better protect women who are generally at higher risk and are more vulnerable to STIs., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Wound care is a serious healthcare concern, often complicated by prolonged inflammation and bacterial infection, which contributes significantly to mortality and morbidity. Agents commonly used to treat chronic wound infections are limited due to toxicity of the therapy, multifactorial etiology of chronic wounds, deep skin infections, lack of sustained controlled delivery of drugs, and development of drug resistance. LL37 is an endogenous host defense peptide possessing antimicrobial activity and is involved in the modulation of wound healing. Serpin A1 (A1) is an elastase inhibitor and has been shown to demonstrate wound-healing properties. Hence, our goal was to develop a topical combination nanomedicine for the controlled sustained delivery of LL37 and A1 at precise synergistic ratio combinations that will significantly promote wound closure, reduce bacterial contamination, and enhance anti-inflammatory activity. We have successfully developed the first solid lipid nanoparticle (SLN) formulation that can simultaneously deliver LL37 and A1 at specific ratios resulting in accelerated wound healing by promoting wound closure in BJ fibroblast cells and keratinocytes as well as synergistically enhancing antibacterial activity against S. aureus and E. coli in comparison to LL37 or A1 alone.

The failures of several first-generation and second-generation small molecule drug-based anti-HIV therapies in various stages of clinical trials are an indication that there is a need for a paradigm shift in the future designs of anti-HIV therapeutics. Over the past several decades, various anti-HIV drugs have been developed, among them, protein/peptide-based therapies. From the first peptide discovered (SJ2176) to the first peptide approved by the Food and Drug Administration (DP178/T20/enfuvirtide/Fuzeon®), anti-HIV proteins/peptides as fusion/entry inhibitors have been shown to provide potent effects and benefits. This review summarizes the past and current endeavors in this area, discusses the potential mechanisms of action for various anti-HIV proteins/peptides, compares the advantages and disadvantages between the different proteins/peptides, and finally, examines the future direction of the field, specifically, strategies that will enhance the therapeutic efficacy of fusion/entry inhibitor-based anti-HIV proteins/peptides. Although there are numerous reviews highlighting the general field of entry/fusion inhibitors, there is a lack of literature focused on protein/peptide-based entry/fusion inhibitors for HIV therapy, and as a result, this review is intended to fill this void by summarizing the past, current, and future development of these macromolecules. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

The use of polymeric devices for controlled sustained delivery of drugs is a promising approach for the prevention of HIV-1 infection. Unfortunately, certain microbicides, when topically applied vaginally, may be cytotoxic to vaginal epithelial cells and the protective microflora present within the female genital tract. In this study, we evaluated the impact of hydroxychloroquine (HCQ)-loaded, reservoir-type, polyurethane intravaginal rings (IVRs) on the growth of Lactobacillus crispatus and Lactobacillus jensenii and on the viability of vaginal and ectocervical epithelial cells. The IVRs were fabricated using hot-melt injection molding and were capable of providing controlled release of HCQ for 24 days, with mean daily release rates of 17.01 ± 3.6 μg/ml in sodium acetate buffer (pH 4) and 29.45 ± 4.84 μg/ml in MRS broth (pH 6.2). Drug-free IVRs and the released HCQ had no significant effects on bacterial growth or the viability of vaginal or ectocervical epithelial cells. Furthermore, there was no significant impact on the integrity of vaginal epithelial cell monolayers, in comparison with controls, as measured by transepithelial electrical resistance. Overall, this is the first study to evaluate the effects of HCQ-loaded IVRs on the growth of vaginal flora and the integrity of vaginal epithelial cell monolayers., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The goal of this study was to develop and characterize a novel intravaginal film platform for targeted delivery of small interfering RNA (siRNA)-loaded nanoparticles (NP) to dendritic cells as a potential gene therapy for the prevention of sexually transmitted human immunodeficiency virus (HIV) infection. Poly(ethylene glycol) (PEG)-functionalized poly(D, L-lactic-co-glycolic acid) (PLGA)/polyethylenimine (PEI)/siRNA NP (siRNA-NP) were fabricated using a modified emulsion-solvent evaporation method and characterized for particle size, zeta potential, encapsulation efficiency (EE), and siRNA release. siRNA-NP were decorated with anti-HLA-DR antibody (siRNA-NP-Ab) for targeting delivery to HLA-DR+ dendritic cells (DCs) and homogeneously dispersed in a biodegradable film consisting of poly vinyl alcohol (PVA) and λ-carrageenan. The siRNA-NP-Ab-loaded film (siRNA-NP-Ab-film) was transparent, displayed suitable physicomechanical properties, and was noncytotoxic. Targeting activity was evaluated in a mucosal coculture model consisting of a vaginal epithelial monolayer (VK2/E6E7 cells) and differentiated KG-1 cells (HLA-DR+ DCs). siRNA-NP-Ab were rapidly released from the film and were able to penetrate the epithelial layer to be taken up by differentiated KG-1 cells. siRNA-NP-Ab demonstrated higher targeting activity and significantly higher knockdown of synaptosome-associated 23-kDa protein (SNAP-23) mRNA and protein when compared to siRNA-NP without antibody conjugation. Overall, these data suggest that our novel siRNA-NP-Ab-film may be a promising platform for preventing HIV infection within the female genital tract.

Multicomponent nutraceuticals are becoming increasingly popular treatments or adjunctive therapies for osteoarthritis in veterinary medicine despite lack of evidence of efficacy for many products. The objective of this study was to evaluate the anti-inflammatory and antioxidant activities of a commercially available C-phycocyanin-based nutraceutical and select constituent ingredients in an in-vitro model of canine osteoarthritis. Normal canine articular chondrocytes were used in an in-vitro model of osteoarthritis. Inflammatory conditions were induced using interleukin-1β. The nutraceutical preparation as a whole, its individual constituents, as well as carprofen were evaluated at concentrations of 0 to 250 μg/mL for reduction of the following inflammatory mediators and indicators of catabolism of the extracellular matrix: prostaglandin E2 (PGE2), tumor necrosis factor-α (TFN-α), interleukin-6 (IL-6), metalloproteinase-3 (MMP-3), nitric oxide, and sulfated glycosaminoglycans (sGAGs). Validated, commercially available assay kits were used for quantitation of inflammatory mediators. The antioxidant capacities, as well as cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and lipoxygenase (LOX) inhibitory activities of the whole nutraceutical preparation and select constituents, were also assessed using validated commercially available assay kits. The antioxidant capacity of the nutraceutical and constituents was concentration-dependent. The nutraceutical and constituents appear to display anti-inflammatory activity primarily through the inhibition of COX-2. The nutraceutical displayed similar strength to carprofen in reducing TNF-α, IL-6, MMP-3, nitric oxide, and sGAGs at select concentration ranges. The C-phycocyanin (CPC)-based nutraceutical and constituents may be able to mediate 3 primary pathogenic mechanisms of osteoarthritis: inflammation, chondral degeneration, and oxidative stress in vitro. The nutraceutical may be clinically useful in veterinary medicine and its efficacy should be further investigated in vivo.

Recently, a growing number of macromolecules such as peptides and proteins have been formulated into various microbicide formulations for the prevention of sexually transmitted infections. However, a fast and reliable high-throughput method for quantitating peptide/protein in polymer-based microbicide formulations is still lacking. As a result, we developed and validated a reversed-phase high-performance liquid chromatography method for the quantitation of gp120 fragment and LL-37 simultaneously in various microbicide gel formulations. This method was capable of detecting a limit of linearity (regression coefficient of 0.999) for gp120 fragment and LL-37 within a range of 0.625-80 and 1.25-80 µg mL -1 , respectively. The lower limit of quantification for gp120 fragment and LL-37 was 1.14 and 0.31 µg mL -1 , respectively. Method validation demonstrated acceptable intra- and inter-day RSD % (<5 %) and accuracy (95.67-100.5 %). Formulating both peptides into polymeric pharmaceutical gel formulations showed high extraction efficiency (in the range of 95.90 ± 3.03 to 111.45 ± 2.51 %). Using this method, we were able to separate and identify the forced degraded products from both peptides simultaneously without affecting the quantitation of both peptides in the polymeric dosage forms. Furthermore, this method was able to detect and separate degradants that were unable to be revealed using gel eletrophoresis.

The vaginal tract is a suitable site for the administration of both local and systemic acting drugs. There are numerous vaginal products on the market such as those approved for contraception, treatment of yeast infection, hormonal replacement therapy, and feminine hygiene. Despite the potential in drug delivery, the vagina is a complex and dynamic organ that requires greater understanding. The recent discovery that injections of double stranded RNA (dsRNA) in Caenorhabditis elegans (C. elegans) results in potent gene specific silencing, was a major scientific revolution. This phenomenon known as RNA interference (RNAi), is believed to protect host genome against invasion by mobile genetic elements such as transposons and viruses. Gene silencing or RNAi has opened new potential opportunities to study the function of a gene in an organism. Furthermore, its therapeutic potential is being investigated in the field of sexually transmitted infections such as human immunodeficiency virus (HIV) and other diseases such as age-related macular degeneration (AMD), diabetes, hypercholesterolemia, respiratory disease, and cancer. This review will focus on the therapeutic potential of siRNA for the treatment and/or prevention of infectious diseases such as HIV, HPV, and HSV within the vaginal tract. Specifically, formulation design parameters to improve siRNA stability and therapeutic efficacy in the vaginal tract will be discussed along with challenges, advancements, and future directions of the field., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Polyethylene glycol (PEG) has been used widely in liposomal formulations as a strategy to inhibit opsonization by plasma proteins and to prolong liposome plasma circulation time. PEG can be incorporated onto the surface of liposomes either during the spontaneous self-assembling process or inserted after vesicle formation. The advantages of employing the PEG postinsertion method include improved drug encapsulation efficiency and the ability to incorporate PEG conjugates for enhanced cell binding and uptake. In this study, we propose to evaluate a cationic lipid nanoparticle formulation containing two PEGylation steps: pre- and post-siRNA insertion. Our results indicate that formulations consisting of the extra PEG post-insertion step significantly increased siRNA circulation in the plasma by two-folds in comparison with the formulations consisting of only the single PEGylation step. Moreover, this formulation was able to efficiently carry siRNA to the tumor site, increase siRNA stability and significantly downregulate luciferase mRNA expression by >50% when compared with the controls in an intraperitoneal and subcutaneous breast cancer tumor model. Overall, our cationic lipid nanoparticle formulation displayed enhanced plasma circulation, reduced liver accumulation, enhanced tumor targeting, and effective gene knockdown--demonstrating excellent utility for the delivery of siRNA., (Copyright © 2012 Wiley Periodicals, Inc.)

Cationic liposomes exhibit a propensity to selectively target tumor-associated blood vessels demonstrating potential value as anti-cancer drug delivery vehicles. Their utility however, is hampered by their biological instability and rapid elimination following i.v. administration. Efforts to circumvent rapid plasma elimination have, to date, focused on decreasing cationic lipid content and incorporating polyethylene glycol (PEG)-modified lipids. In this study we wanted to determine whether highly charged cationic liposomes with surface-associated PEG could be designed to exhibit extended circulation lifetimes, while retaining tumor vascular targeting properties in an HT29 colorectal cancer xenograft model. Cationic liposomes prepared of DSPC, cationic lipids (DODAC, DOTAP, or DC-CHOL), and DSPE-PEG(2000) were studied. Our results demonstrate that formulations prepared with 50 mol% DODAC or DC-CHOL, and 20 mol% DSPE-PEG(2000) exhibited circulation half-lives ranging from 6.5 to 12.5 h. Biodistribution studies demonstrated that DC-CHOL formulations prepared with DSPE-PEG(2000) accumulated threefold higher in s.c. HT29 tumors than its PEG-free counterpart. Fluorescence microscopy studies suggested that the presence of DSPE-PEG(2000) did not adversely affect liposomal tumor vasculature targeting. We show for the first time that it is achievable to design highly charged, highly pegylated (20 mol% DSPE-PEG(2000)) cationic liposomes which exhibit both extended circulation lifetimes and tumor vascular targeting properties., ((c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association)

Endotoxin-induced inflammation alters the hepatic expression of the drug efflux transporter genes mdr1b (Abcb1b) and mrp3 (Abcc3) in rats. In this study, we identified a novel kruppel-like zinc finger protein 6 (KLF6) cis-element on the rat mdr1b promoter which is important for basal activity and IL-1beta and endotoxin-mediated induction in gene transcription. Interestingly, KLF6 also functioned as a negative transcriptional regulator, inhibiting TNF-alpha-mediated induction of mdr1b. Furthermore, novel CCAAT/enhancer binding protein beta (C/EBPbeta) and heat shock factor 4 (HSF4) transcription binding sites were identified on the rat mrp3 promoter. Deletion of the HSF4 element significantly increased transcriptional activity of the mrp3 gene when exposed to TNF-alpha. Endotoxin treatment significantly affected transcriptional activity only in C/EBPbeta and HSF4 double deletion mrp3 promoter constructs. In summary, KLF6 and HSF4 are stimuli-specific regulatory elements which may be important in the control of the rat mdr1b and mrp3 genes during health and disease.

Recently, we developed a novel implantable drug delivery system which can provide sustained intraperitoneal (i.p.) delivery of paclitaxel (PTX). As the impact of local sustained delivery on the development of multidrug resistance (MDR) is unknown, the objective of this study was to determine the impact of this drug delivery system on the in vivo expression of MDR1/P-glycoprotein (PGP) in a human ovarian xenograft tumor model. As compared to controls, intermittent i.p. dosing with PTX formulated in Cremophor EL (PTX(CrEL)) induced a two-fold increase in mRNA levels of MDR1 after a 14-day dosing period. On the other hand, sustained i.p. delivery of PTX with the implant system (PTX(film)) did not significantly affect MDR1 expression. Immunodetection of PGP in isolated xenografts supported the mRNA data. Histological analysis by H&E staining demonstrated a dose-dependent increase in tumor necrosis in the PTX(film) treated animals. Further, in vitro studies in human ovarian carcinoma cells also demonstrated a significant induction in the efflux activity of PGP with intermittent dosing schedules to PTX(CrEL) whereas this was not seen in cells dosed with PTX(film). Our findings suggest that sustained i.p. administration with PTX(film) attenuates development of MDR, suggesting that sustained, localized delivery of chemotherapeutic agents may improve current treatment strategies for ovarian cancer.

DNA frayed wires (DNA(FW)) are an alternate form of DNA organization formed by the self-association of several strands of guanine-rich oligonucleotides. The purpose of this study was to define for the first time the blood clearance kinetics, tissue distribution, and stability of DNA(FW) in vivo in mice. Single bolus doses (1200 pmol/mouse) of (32)P-DNA(FW) and (32)P-random DNA were administered intravenously (IV) and intraperitoneally (IP) followed by scheduled blood, urine, fecal and tissue samplings. Blood clearance kinetics was described well by a first order two-compartment open model. The overall half-lives of elimination from the central compartment (T(1/2))(K10) were 3.57+/-0.1h for IV and 2.38+/-0.11 h for IP. In contrast, random DNA was completely degraded after 15 min regardless of the route of administration. Tissue distribution results demonstrated that DNA(FW) were primarily distributed and retained in the liver, intestines, kidneys, and heart. Low levels could also be detected in brain. Autoradiographs of blood, tissues, feces and urine extracts established that DNA(FW) remained intact after administration as no measurable levels of metabolites or degradation products were found after 24h. (32)P-DNA(FW) was primarily eliminated via hepato-biliary excretion into feces after either IV or IP administration (51.8+/-4.53% and 36.2+/-3.4%, respectively). The improved stability and longer half-life of DNA(FW), previously shown in vitro, is also seen in vivo, indicating that DNA(FW) may provide a stable delivery system for DNA gene therapies. In conclusion, this is the first study demonstrating the in vivo stability, pharmacokinetics, and disposition of DNA superstructures.

Various mechanisms have been implicated in the development of resistance of cancer cells to chemotherapy. Multidrug resistance (MDR) is a phenomenon in which cancer cells are resistant to the cytotoxic effects of various structurally and mechanistically unrelated chemotherapeutic agents. One major mechanism by which this occurs is through the over-expression of ATP-dependent drug efflux transporters such as the P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP). Regulation of MDR can occur at many levels including transcriptional, mRNA, protein and post-translational. In recent years it has been demonstrated that alterations in the expression and activity of the MDR transporters are seen in numerous tissues during an inflammatory response. An acute inflammatory response is associated with many conditions including infection, injury, hypoxia and stress and is known to result in the induction of several pro-inflammatory cytokines. Whether the function of cytokines can be harnessed in overcoming drug resistance of tumors has yet to be examined and explored. In this review, we will focus on the various studies investigating the regulation of MDR during an inflammatory response, in particular by cytokines. The mediators and pathways involved as well as the possible mechanisms of MDR regulation will be discussed. It is hoped that by understanding the clinical importance of inflammatory mediators in MDR, new doors will open and future insights will lead to the development of novel immunotherapeutics for the treatment of cancer.

Recently, a novel chitosan-based implantable formulation (chitosan-ePC) was developed to provide controlled, local release of paclitaxel (PTX) for the treatment of ovarian tumors. Hence, the objective of this study was to evaluate this delivery system in vitro in human ovarian SKOV-3 cells and in vivo in mice with intraperitoneal implants of drug-free or 14C-PTX-chitosan-ePC films. In vitro, 14C-PTX-chitosan-ePC implants (10 mg) provided zero-order constant release of 0.92+/-0.03 pg/day PTX over 5 days. Released PTX retained dose-dependent activity; effectively inhibiting SKOV-3 proliferation with an ED50 of 211 ng/ml of released PTX. Drug-free implants did not affect cell viability or cell morphology of SKOV-3 cells. A sustained, zero-order release of PTX was also seen in vivo over a 2 week period in mice implanted with 14C-PTX-chitosan-ePC films. Correlations between the in vitro and in vivo release of PTX was highly significant (R2 = 0.975). After 2-4 weeks, mice with chitosan-ePC implants did not demonstrate any signs of encapsulation, inflammation or infection. Overall, our in vitro and in vivo results demonstrated zero-order drug release and biocompatibility of the novel chitosan-ePC film. This indicates potential usefulness of chitosan-ePC implants in the sustained and local delivery of anti-neoplastic agents.