The Encyclopedia of Biomedical Polymers & Polymeric Biomaterials presents state-of-the-art research and development on the synthesis, properties, and applications of novel polymers in a vital area. This groundbreaking work includes an extremely large number of contributors for a reference publication in biomedical polymers and polymeric biomaterials. With multiple articles on many subjects, the encyclopedia offers a broad-based perspective on a multitude of topics, as well as detailed research information, figures, tables, illustrations, and references.
Current nanostructured biomaterials-based drug delivery vehicles for bone regeneration applications often show suboptimal cellular uptake and inferior drug loading. To overcome these challenges, we have developed a biomimetic cell-derived nanoparticle (CDN) loaded with the Food and Drug Administration-approved small-molecule therapeutic Dexamethasone, to induce osteogenic differentiation in human adipose-derived stem cells. The drug-loaded CDNs were cytocompatible, maintained hydrodynamic stability with uniform spherical shape and size, and exhibited high-percentage drug loading along with rapid cellular uptake and stem cell differentiation. These results demonstrate for the first time the preparation of Dexamethasone-loaded CDNs capable of directing stem cell fate for advanced bone regeneration applications.
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.
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.
Parenteral route (also referred to as injection route) of drug administration has many advantages including rapid systemic efects and the ability to deliver drugs locally to a given region. Unfortunately, the major challenge with parenteral drug administration is low patient acceptance which stems from the fear and pain of needles. As a result, alternative routes of drug administration are desired by most patients. In this special issue, we have highlighted some of the major advancements in the felds of transdermal and mucosal drug delivery and its rapidly growing potential in improving global health.
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.
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..
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.
The technology of 3D printing offers a customizable manufacturing method, while decreasing material waste. The sustainability of this industry is important as it grows into a more important role in the manufacturing market. The use and development of recyclable, biodegradable, and sustainably sourced materials for extrusion‐based 3D printing may allow for a decrease in environmental impacts and demonstrate the importance of this technology as a sustainable method of manufacturing. The goal of this article is to provide an insight into the state of the development of sustainable materials for 3D printing, such as recyclable, biodegradable, and cellulose‐based feedstock.
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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 lipidbilayer. 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.
Intravenous administration of anti-α4β7 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-α4β7 antibody may be safer and more effective. With this in mind, we developed a novel intravaginal formulation consisting of anti-α4β7 monoclonal antibody–conjugated nanoparticles (NPs) loaded in a 1% hydroxyethylcellulose (HEC) gel (NP-α4β7 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 α4β7, and occupied ~40% of α4β7 expressed by these subsets and ~25% of all cells expressing α4β7. Blocking of the α4β7 was restricted to the vaginal tract without any changes detected systemically.
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). 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. VisiblexTM 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 hours 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 membranes and the anionic NPs, and the change in pH-responsive morphology of the membrane. In vitro biocompatibility studies of the membranes 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, these porous pH-responsive PU membranes demonstrated their potential in serving as "window" membranes of reservoir-IVRs for pH-responsive intravaginal release of NPs.
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.
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) and1H nuclear magnetic resonance (1H 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.
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.
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 towards 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.
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.
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.
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 in pH 4.5 and pH 7.0 vaginal fluid simulants (VFSs) to negatively-charged spherically-shaped nanoparticles 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 312 h. 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 the 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.
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.
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), 1H-nuclear magnetic resonance (1H-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 24 h at 37°C and over 20% of total NaDF released at pH 7 within 3 h 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 anionic drug delivery. [STATEMENT OF SIGNIFICANCE]: A reversible and sharp switch between "off" and "on" drug release is achieved for the first time through a new pH-sensitive polyurethane membrane, which can serve as a window membrane in a reservoirtype intravaginal ring 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 softsegment of polyurethane. This polyurethane biomaterial holds great potential to better protect women who are generally at higher risk and are more vulnerable to STIs.
Wound treatment remains one of the most prevalent and economically burdensome healthcare concerns, often complicated by prolonged inflammation and bacterial infection, contributing to morbidity and mortality. 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 that has been shown to exhibit 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 use of polymeric devices for the controlled sustained delivery of drugs is a promising approach for the prevention of HIV-1 infection. Unfortunately, certain microbicides when applied topically 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 a hydroxychloroquine (HCQ)-loaded reservoir-type polyurethane intravaginal ring (IVR) on the growth of Lactobacillus crispatus and Lactobacillus jensenii, and on the viability of vaginal and ectocervical epithelial cells. The IVR was fabricated using hot-melt injection molding and was capable of providing controlled release of HCQ for 24 days with a mean daily release 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 impact on bacterial growth or the viability of vaginal and ectocervical epithelial cells. Furthermore, there was no significant impact on vaginal epithelial cell monolayer integrity in comparison to controls as measured by transepithelial electrical resistance. Overall, this is the first study to evaluate the impact of HCQ-loaded IVRs on the growth of vaginal flora and the monolayer integrity of vaginal epithelial cells.
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.
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. NPs 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 physico-mechanical properties, and was non-cytotoxic. Targeting activity was evaluated in a mucosal co-culture 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 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, this data suggest that our novel siRNA-NP-Ab-film may be a promising platform for preventing HIV infection within the female genital tract.
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.
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.
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.
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.
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.
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.
Localized and sustained delivery of anti-cancer agents to the tumor site has great potential for the treatment of solid tumors. A chitosan–egg phosphatidylcholine (chitosan–ePC) implant system containing PLA-b-PEG/PLA nanoparticles has been developed for the delivery of paclitaxel to treat ovarian cancer. Production of volumes of ascites fluid in the peritoneal cavity is a physical manifestation of ovarian cancer. In vitro release studies of paclitaxel from the implant were conducted in various fluids including human ascites fluid. A strong correlation (r2 = 0.977) was found between the release of paclitaxel in ascites fluid and PBS containing lysozyme (pH 7.4) at 37 °C. The drug release mechanism for this system was proposed based on swelling, degradation and morphology data. In addition, in vitro release of paclitaxel was found to be a good indicator of the in vivo release profile (correlation between release rates: r2 = 0.965). Release of paclitaxel was found to be sustained over a four-week period following implantation of the chitosan–ePC system into the peritoneal cavity of healthy Balb/C mice. Also, the concentrations of paclitaxel in both plasma and tissues (e.g. liver, kidney and small intestine) were found to be relatively constant.
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.
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