Engineered albumin-functionalized nanoparticles for improved FcRn binding enhance oral delivery of insulin.

Oral delivery of biopharmaceuticals, as insulin, is hampered by rapid degradation and inefficient absorption in the gastrointestinal tract (GIT). To solve this, a new class of biodegradable poly(lactic-co-glycolic)-poly(ethylene glycol) (PLGA-PEG) mucodiffusive nanoparticles (NPs) was designed. Specifically, these were decorated with site-specific conjugated human albumin, engineered for improved pH dependent binding to the neonatal Fc receptor (FcRn), which naturally mediates transport of albumin across the intestinal epithelium. The designed NPs of monodisperse 150 nm in size were 10% loaded with insulin and their surface was successfully functionalized with human albumin. Importantly, the engineered albumin-functionalized NPs bound human FcRn favorably in a pH dependent manner and showed enhanced transport across polarized cell layers. When orally administered to human FcRn expressing mice induced with diabetes, a reduction of glycemia was measured as a function of receptor targeting, with up to around 40% reduction after 1 h post-delivery. Thus, biodegradable PLGA-PEG NPs decorated with human albumin for improved FcRn-dependent transport offer a novel attractive strategy for delivery of encapsulated biopharmaceuticals across intestinal barriers.

Colorectal distribution and retention of polymeric nanoparticles following incorporation into a thermosensitive enema.

Nanotechnology-based systems have been proposed for rectal drug delivery, often rendering promising outcomes concerning disease prophylaxis or therapeutics. However, nanocarriers often feature reduced colorectal retention when administered in liquid vehicles (enemas). Semi-solid platforms may be considered as alternative but usually result in limited local distribution. Thermosensitive enemas undergoing sol-gel transition just below body temperature have been used for abbreviating these issues, but the actual impact on the colorectal distribution and retention of incorporated nanosystems is not clear. We prepared and characterized a potential drug delivery platform by incorporating poly(lactic-co-glycolic acid)-based nanoparticles (170-180 nm mean hydrodynamic diameter) into a poloxamer 407-based thermosensitive enema (NPs-in-thermo). The system featured suitable functional properties for rectal administration such as sol-gel transition temperature of approximately 27-28 °C, sol-gel transition time of 1.6 min, and viscosity around 31 and 2100 mPa s at 20 °C and 37 °C, respectively. NPs-in-thermo presented osmolality and pH values deemed compatible with the colorectal compartment, as well as reduced toxicity to the Caco-2 colorectal cell line. The composite system was also used to incorporate the anti-HIV microbicide model drug dapivirine. In vitro studies showed that dapivirine-loaded NPs-in-thermo was able to provide overall faster drug release as compared to dapivirine directly dispersed into phosphate buffered saline or the thermosensitive enema base. Finally, NPs-in-thermo was tested for distribution and retention in a mouse model by in vivo and ex vivo near infrared imaging. Qualitative and semi-quantitative data indicated that NPs exhibited slower but overall wider distribution and enhanced retention in the distal colon of mice treated intrarectally with NPs-in-thermo, namely when compared to NPs dispersed in liquid phosphate buffered saline. Overall, our data support that thermosensitive enemas may provide suitable platforms for the rectal administration of polymeric NPs, namely in the context of drug delivery.

Using microfluidic platforms to develop CNS-targeted polymeric nanoparticles for HIV therapy.

The human immunodeficiency virus (HIV) uses the brain as reservoir, which turns it as a promising target to fight this pathology. Nanoparticles (NPs) of poly(lactic-co-glycolic) acid (PLGA) are potential carriers of anti-HIV drugs to the brain, since most of these antiretrovirals, as efavirenz (EFV), cannot surpass the blood-brain barrier (BBB). Forasmuch as the conventional production methods lack precise control over the final properties of particles, microfluidics emerged as a prospective alternative. This study aimed at developing EFV-loaded PLGA NPs through a conventional and microfluidic method, targeted to the BBB, in order to treat HIV neuropathology. Compared to the conventional method, NPs produced through microfluidics presented reduced size (73 nm versus 133 nm), comparable polydispersity (around 0.090), less negative zeta-potential (-14.1 mV versus -28.0 mV), higher EFV association efficiency (80.7% versus 32.7%) and higher drug loading (10.8% versus 3.2%). The microfluidics-produced NPs also demonstrated a sustained in vitro EFV release (50% released within the first 24 h). NPs functionalization with a transferrin receptor-binding peptide, envisaging BBB targeting, proved to be effective concerning nuclear magnetic resonance analysis (δ = -0.008 ppm; δ = -0.017 ppm). NPs demonstrated to be safe to BBB endothelial and neuron cells (metabolic activity above 70%), as well as non-hemolytic (1-2% of hemolysis, no morphological alterations on erythrocytes). Finally, functionalized nanosystems were able to interact more efficiently with BBB cells, and permeability of EFV associated with NPs through a BBB in vitro model was around 1.3-fold higher than the free drug.

Surface modification with polyethylene glycol enhances colorectal distribution and retention of nanoparticles.

Dense surface modification with short chain polyethylene glycol (PEG) has been previously demonstrated as favoring the transport of nanoparticles (NPs) across mucus. However, the ability of such approach to influence the distribution and retention of NPs along the length of the colorectum after rectal delivery has not been previously established. Herein, the distribution and retention of poly(lactic-co-glycolic acid) NPs modified with PEG in a non-covalent fashion are reckoned in a mouse model. Despite overall rapid depletion, both PEG-modified and non-modified NPs are able to reach the middle segment of the colon. PEG-modified NPs are able to enhance retention up to at least two hours post-administration, contrasting with nearly residual levels observed for non-modified NPs after 15 min. The ability of PEG-modified NPs to putatively cross mucus also appears to promote association with tissues. Overall, the work provides significant insights as to the behavior of NPs in the colorectum, which could be valuable for the development of rectal nanomedicines. It further reinforces the potential usefulness of PEG-modified NPs as mucus-penetrating carriers for mucosal drug delivery.

Noncovalent PEG Coating of Nanoparticle Drug Carriers Improves the Local Pharmacokinetics of Rectal Anti-HIV Microbicides.

Antiretroviral drug nanocarriers hold great promise for developing anti-human immunodeficiency virus (HIV) rectal microbicides. However, challenges remain, namely, concerning which properties are more suited for enhancing colorectal distribution and retention of microbicide compounds. In this work, we developed and assessed the in vitro and in vivo performance of poly(lactic- co-glycolic acid) (PLGA)-based nanoparticles (NPs) as carriers for the model drug efavirenz (EFV). We particularly focused on the effect of noncovalent poly(ethylene glycol) coating of PLGA NPs (PEG-PLGA NPs) conferring a mucus-diffusive behavior on the pharmacokinetics (PK) of EFV following rectal administration to mice. Drug-loaded PLGA NPs and PEG-PLGA NPs (200-225 nm) were obtained by nanoprecipitation. Both types of systems were able to retain native antiretroviral activity of EFV in vitro, while featuring lower cytotoxicity against different epithelial cell lines and HIV target cells. Also, PLGA NPs and PEG-PLGA NPs were readily taken up by colorectal cell lines and mildly reduced EFV permeation while increasing membrane retention in Caco-2 and Caco-2/HT29-MTX cell monolayer models. When administered intrarectally to CD-1 mice in phosphate-buffered saline (pH 7.4), EFV-loaded PEG-PLGA NPs consistently provided higher drug levels in colorectal tissues and lavages, as compared to free EFV or drug-loaded PLGA NPs. Mean values for the area-under-the-curve between 15 min and 12 h following administration were particularly higher for PEG-PLGA NPs in distal and middle colorectal tissues, with relative bioavailability values of 3.7 and 29, respectively, as compared to free EFV (2.2 and 6.0 over PLGA NPs, respectively). Systemic exposure to EFV was reduced for all treatments. NPs were further shown safe after once-daily administration for 14 days, as assessed by histological analysis of colorectal tissues and chemokine/cytokine assay of rectal lavages. Overall, PEG-PLGA NPs demonstrated to be safe carriers for rectal microbicide drug delivery and able to provide enhanced local PK that could be valuable in preventing rectal HIV transmission.

PEGylated PLGA Nanoparticles As a Smart Carrier to Increase the Cellular Uptake of a Coumarin-Based Monoamine Oxidase B Inhibitor.

Despite research efforts to discover new drugs for Parkinson treatment, the majority of candidates fail in preclinical and clinical trials due to inadequate pharmacokinetic properties, namely blood-brain barrier permeability. Within the high demand to introduce new drugs to market, nanotechnology can be used as a solution. Accordingly, PEGylated PLGA nanoparticles (NPs) were used as a smart delivery carrier to solve the suboptimal aqueous solubility, which precludes its use in in vivo assays, of a potent, reversible, and selective monoamine oxidase B inhibitor (IMAO-B) (coumarin C75, IC50 = 28.89 ± 1.18 nM). Long-term stable PLGA@C75 NPs were obtained by nanoprecipitation method, with sizes around 105 nm and a zeta potential of -10.1 mV. The encapsulation efficacy was around 50%, achieving the final C75 concentration of 807 ± 30 µM in the nanoformulation, which corresponds to a therapeutic concentration 27828-fold higher than its IC50 value. Coumarin C75 showed cytotoxic effects at 50 µM after 48 and 72 h of exposure in SH-SY5Y, Caco-2, and hCMEC/D3 cell lines. Remarkably, no cytotoxic effects were observed after nanoencapsulation. Furthermore, the data obtained from the P-gp-Glo assay and the cellular uptake studies showed that C75 is a P-glycoprotein (P-gp) substrate having a lower uptake profile in intestinal and brain endothelial cells. Moreover, it was shown that this membrane transporter influences C75 permeability profile in Caco-2 and hCMEC/D3 cells. Interestingly, PLGA NPs inhibited P-gp and were able to cross intestinal and brain membranes allowing the successful transport of C75 through this type of biological barriers. Overall, this work showed that nanotechnology can be used to solve drug discovery related drawbacks.

Development and in vivo safety assessment of tenofovir-loaded nanoparticles-in-film as a novel vaginal microbicide delivery system.

UNLABELLED: Topical pre-exposure prophylaxis (PrEP) with antiretroviral drugs holds promise in preventing vaginal transmission of HIV. However, significant biomedical and social issues found in multiple past clinical trials still need to be addressed in order to optimize protection and users' adherence. One approach may be the development of improved microbicide products. A novel delivery platform comprising drug-loaded nanoparticles (NPs) incorporated into a thin polymeric film base (NPs-in-film) was developed in order to allow the vaginal administration of the microbicide drug candidate tenofovir. The system was optimized for relevant physicochemical features and characterized for biological properties, namely cytotoxicity and safety in a mouse model. Tenofovir-loaded poly(lactic-co-glycolic acid) (PLGA)/stearylamine (SA) composite NPs with mean diameter of 127nm were obtained with drug association efficiency above 50%, and further incorporated into an approximately 115µm thick, hydroxypropyl methylcellulose/poly(vinyl alcohol)-based film. The system was shown to possess suitable mechanical properties for vaginal administration and to quickly disintegrate in approximately 9min upon contact with a simulated vaginal fluid (SVF). The original osmolarity and pH of SVF was not affected by the film. Tenofovir was also released in a biphasic fashion (around 30% of the drug in 15min, followed by sustained release up to 24h). The incorporation of NPs further improved the adhesive potential of the film to ex vivo pig vaginal mucosa. Cytotoxicity of NPs and film was significantly increased by the incorporation of SA, but remained at levels considered tolerable for vaginal delivery of tenofovir. Moreover, histological analysis of genital tissues and cytokine/chemokine levels in vaginal lavages upon 14days of daily vaginal administration to mice confirmed that tenofovir-loaded NPs-in-film was safe and did not induce any apparent histological changes or pro-inflammatory response. Overall, obtained data support that the proposed delivery system combining the use of polymeric NPs and a film base may constitute an exciting alternative for the vaginal administration of microbicide drugs in the context of topical PrEP. STATEMENT OF SIGNIFICANCE: The development of nanotechnology-based microbicides is a recent but promising research field seeking for new strategies to circumvent HIV sexual transmission. Different reports detail on the multiple potential advantages of using drug nanocarriers for such purpose. However, one important issue being frequently neglected regards the development of vehicles for the administration of microbicide nanosystems. In this study, we propose and detail on the development of a nanoparticle-in-film system for the vaginal delivery of the microbicide drug candidate tenofovir. This is an innovative approach that, to our best knowledge, had never been tested for tenofovir. Results, including those from in vivo testing, sustain that the proposed system is safe and holds potential for further development as a vaginal microbicide product.

Pharmacological and toxicological assessment of innovative self-assembled polymeric micelles as powders for insulin pulmonary delivery.

AIM: Explore the use of polymeric micelles in the development of powders intended for pulmonary delivery of biopharmaceuticals, using insulin as a model protein. MATERIALS & METHODS: Formulations were assessed in vitro for aerosolization properties and in vivo for efficacy and safety using a streptozotocin-induced diabetic rat model. RESULTS: Powders presented good aerosolization properties like fine particle fraction superior to 40% and a mass median aerodynamic diameter inferior of 6 µm. Endotracheally instilled powders have shown a faster onset of action than subcutaneous administration of insulin at a dose of 10 IU/kg, with pharmacological availabilities up to 32.5% of those achieved by subcutaneous route. Additionally, micelles improved the hypoglycemic effect of insulin. Bronchoalveolar lavage screening for toxicity markers (e.g., lactate dehydrogenase, cytokines) revealed no signs of lung inflammation and cytotoxicity 14 days postadministration. CONCLUSION: Developed powders showed promising safety and efficacy characteristics for the systemic delivery of insulin by pulmonary administration.

Nanoparticles-in-film for the combined vaginal delivery of anti-HIV microbicide drugs.

Combining two or more antiretroviral drugs in one medical product is an interesting but challenging strategy for developing topical anti-HIV microbicides. We developed a new vaginal delivery system comprising the incorporation of nanoparticles (NPs) into a polymeric film base - NPs-in-film - and tested its ability to deliver tenofovir (TFV) and efavirenz (EFV). EFV-loaded poly(lactic-co-glycolic acid) NPs were incorporated alongside free TFV into fast dissolving films during film manufacturing. The delivery system was characterized for physicochemical properties, as well as genital distribution, local and systemic 24h pharmacokinetics (PK), and safety upon intravaginal administration to mice. NPs-in-film presented suitable technological, mechanical and cytotoxicity features for vaginal use. Retention of NPs in vivo was enhanced both in vaginal lavages and tissue when associated to film. PK data evidenced that vaginal drug levels rapidly decreased after administration but NPs-in-film were still able to enhance drug concentrations of EFV. Obtained values for area-under-the-curve for EFV were around one log10 higher than those for the free drugs in aqueous vehicle (phosphate buffered saline). Film alone also contributed to higher and more prolonged local drug levels as compared to the administration of TFV and EFV in aqueous vehicle. Systemic exposure to both drugs was low. NPs-in-film was found to be safe upon once daily vaginal administration to mice, with no significant genital histological changes or major alterations in cytokine/chemokine profiles being observed. Overall, the proposed NPs-in-film system seems to be an interesting delivery platform for developing combination vaginal anti-HIV microbicides.