Platforms to test the temporospatial capabilities of carrier systems in delivering growth factors to benefit vascular bioengineering.

In this study we produced a set of in vitro culture platforms to model vascular cell responses to growth factors and factor delivery vehicles. Two of the systems (whole vessel and whole lung vascular development) were supported by microfluidic systems facilitating media circulation and waste removal. We assessed vascular endothelial growth factor (VEGF) delivery by Pluronic F-127 hydrogel, 30 nm pore-sized microparticles (MPs), 60 nm pore-sized MP or a 50/50 mixture of 30 and 60 nm pore-sized MP. VEGF was delivered to porcine acellular lung vascular scaffolds (2.5 cm2 square pieces or whole 3D segments of acellular blood vessels) as well as whole acellular lung scaffolds. Scaffold-cell attachment was examined as was vascular tissue formation. We showed that a 50/50 mixture of 30 and 60 nm pore-sized silicon wafer MPs allowed for long-term release of VEGF within the scaffold vasculature and supported vascular endothelial tissue development during in vitro culture.

Spray-Dried Nanoparticle-in-Microparticle Delivery Systems (NiMDS) for Gene Delivery, Comprising Polyethylenimine (PEI)-Based Nanoparticles in a Poly(Vinyl Alcohol) Matrix.

Nucleic acid-based therapies rely on efficient formulations for nucleic acid protection and delivery. As nonviral strategies, polymeric and lipid-based nanoparticles have been introduced; however, biological efficacy and biocompatibility as well as poor storage properties due to colloidal instability and their unavailability as ready-to-use systems are still major issues. Polyethylenimine is the most widely explored and promising candidate for gene delivery. Polyethylenimine-based polyplexes and their combination with liposomes, lipopolyplexes, are efficient for DNA or siRNA delivery in vitro and in vivo. In this study, a highly potent spray-dried nanoparticle-in-microparticle delivery system is presented for the encapsulation of polyethylenimine-based polyplexes and lipopolyplexes into poly(vinyl alcohol) microparticles, without requiring additional stabilizing agents. This easy-to-handle gene delivery device allows prolonged nanoparticle storage and protection at ambient temperature. Biological analyses reveal further advantages regarding profoundly reduced cytotoxicity and enhanced transfection efficacies of polyethylenimine-based nanoparticles from the nanoparticle-in-microparticle delivery system over their freshly prepared counterparts, as determined in various cell lines. Importantly, this nanoparticle-in-microparticle delivery system is demonstrated as ready-to-use dry powder to be an efficient device for the inhalative delivery of polyethylenimine-based lipopolyplexes in vivo, as shown by transgene expression in mice after only one administration.

Multidomain drug delivery systems of ß-casein micelles for the local oral co-administration of antiretroviral combinations.

The antiretroviral (ARV) cocktailrevolved the treatment of the human immunodeficiency virus (HIV) infection. Drug combinations have been also tested to treat other infectious diseases, including the recentcoronavirus disease 2019 (COVID-19) outbreak. To simplify administration fixed-dose combinationshave been introduced, however, oral anti-HIV therapy still struggles with low oral bioavailability of many ARVs.This work investigated the co-encapsulation of two clinically relevant ARV combinations,tipranavir (TPV):efavirenz (EFV) anddarunavir (DRV):efavirenz (EFV):ritonavir (RTV),within the core of ß-casein (bCN) micelles. Encapsulation efficiency in both systems was ~100%. Cryo-transmission electron microscopy and dynamic light scattering of the ARV-loaded colloidaldispersions indicatefull preservation of the spherical morphology, and x-ray diffraction confirm that the encapsulated drugs are amorphous. To prolong the physicochemical stabilitythe formulations were freeze-driedwithout cryo/lyoprotectant, and successfully redispersed, with minor changes in morphology.Then, theARV-loaded micelles were encapsulated within microparticles of Eudragit® L100, which prevented enzymatic degradation and minimized drug release under gastric-like pH conditionsin vitro. At intestinal pH, the coating polymer dissolved and released the nanocarriers and content. Overall, our results confirm the promise of this flexible and modular technology platform for oral delivery of fixed dose combinations.

Nanoparticle-in-microparticle oral drug delivery system of a clinically relevant darunavir/ritonavir antiretroviral combination.

Nanonizationhas been extensively investigated to increase theoral bioavailability of hydrophobicdrugsin general andantiretrovirals(ARVs)used inthe therapy of the human immunodeficiency virus (HIV) infection in particular. Weanticipatedthatin the caseofprotease inhibitors, a family of pH-dependent ARVsthatdisplay high aqueous solubility undertheacidconditionsof thestomach andextremely low solubilityunder the neutral ones ofthe small intestine, this strategy might failowing to an uncontrolled dissolution-re-precipitation process that will take place along the gastrointestinal tract.To tackle thisbiopharmaceutical challenge, in this work, wedesigned, produced and fully characterized a novelNanoparticle-in-MicroparticleDelivery System(NiMDS)comprised of pure nanoparticlesofthefirst-line protease inhibitor darunavir(DRV) and itsboosting agentritonavir (RIT) encapsulated within film-coated microparticles.For this, a clinically relevant combination of pure DRV and RIT nanoparticles wassynthesized by a sequential nanoprecipitation/solvent diffusion and evaporation method employing sodium alginateas viscosity stabilizer. Then, pure nanoparticles were encapsulated within calcium alginate/chitosanmicroparticlesthat were film-coated with a series ofpoly(methacrylate) copolymers with differential solubility in the gastrointestinal tract. This coating ensured full stability under gastric-like pH and sustained drug release under intestinal one. PharmacokineticstudiesconductedinalbinoSpragueDawleyratsshowed that DRV/RIT-loadedNiMDSs containing 17% w/w drug loading based on dry weight significantlyincreasedthe oral bioavailabilityof DRVby 2.3-foldwith respect to both theunprocessedandthenanonized DRV/RIT combinations that showed statistically similar performance. Moreover, they highlighted the limited advantage of only drugnanonizationto improve the oral pharmacokinetics of protease inhibitors and the potential of our novel delivery approach to improve the oral pharmacokinetics of nanonized poorly water-soluble drugs displaying pH-dependent solubility. STATEMENT OF SIGNIFICANCE: Protease inhibitors (PIs) are gold-standard drugs in many ARV cocktails. Darunavir (DRV) is the latest approved PI and it is included in the 20th WHO Model List of Essential Medicines. PIs poorly-water soluble at intestinal pH and more soluble under gastric conditions. Drug nanonization represents one of the most common nanotechnology strategies to increase dissolution rate of hydrophobic drugs and thus, their oral bioavailability. For instance, pure drug nanosuspensions became the most clinically relevant nanoformulation. However, according to the physicochemical properties of PIs, nanonization does not appear as a very beneficial strategy due to the fast dissolution rate anticipated under the acid conditions of the stomach and their uncontrolled recrystallization and precipitation in the small intestine that might result in the formation of particles of unpredictable size and structure (e.g., crystallinity and polymorphism) and consequently, unknown dissolution rate and bioavailability. In this work, we developed a sequential nanoprecipitation method for the production of pure nanoparticles of DRV and its boosting agent ritonavir in a clinically relevant 8:1 wt ratio using alginate as viscosity stabilizer and used this nanosuspension to produce a novel kind of nanoparticle-in-microparticle delivery system that was fully characterized and the pharmacokinetics assessed in rats. The most significant points of the current manuscript are.

Improved delivery of PLGA microparticles and microparticle-cell scaffolds in clinical needle gauges using modified viscosity formulations.

Polymer microparticles are widely used as acellular drug delivery platforms in regenerative medicine, and have emerging potential as cellular scaffolds for therapeutic cell delivery. In the clinic, PLGA microparticles are typically administered intramuscularly or subcutaneously, with the clinician and clinical application site determining the precise needle gauge used for delivery. Here, we explored the role of needle diameter in microparticle delivery yield, and develop a modified viscosity formulation to improve microparticle delivery across a range of clinically relevant needle diameters. We have identified an optimal biocompatible formulation containing 0.25% pluronic F127 and 0.25% carboxymethyl cellulose, which can increase delivery payload to 520% across needle gauges 21-30G, and note that needle diameter impacts delivery efficacy. We use this formulation to increase the delivery yield of PLGA microparticles, and separately, PLGA-cell scaffolds supporting viable mesenchymal stem cells (MSCs), demonstrating the first in vitro delivery of this cell scaffold system. Together, these results highlight an optimal formulation for the delivery of microparticle and microparticle-cell scaffolds, and illustrate how careful choice of delivery formulation and needle size can dramatically impact delivery payload.

Defining optimal permeant characteristics for ultrasound-mediated gastrointestinal delivery.

Ultrasound-mediated drug delivery in the gastrointestinal (GI) tract is a bourgeoning area of study. Localized, low-frequency ultrasound has recently been shown to enable significant enhancement in delivery of a broad set of active pharmaceutical ingredients including small molecules, proteins, and nucleic acids without any formulation or encapsulation of the therapeutic. Traditional chemical formulations are typically required to protect, stabilize, and enable the successful delivery of a given therapeutic. The use of ultrasound, however, may make delivery insensitive to the chemical formulation. This might open the door to chemical formulations being developed to address other properties besides the deliverability of a therapeutic. Instead, chemical formulations could potentially be developed to achieve novel pharmacokinetics, without consideration of that particular formulation's ability to penetrate the mucus barrier passively. Here we investigated the effect of permeant size, charge, and the presence of chemical penetration enhancers on delivery to GI tissue using ultrasound. Short ultrasound treatments enabled delivery of large permeants, including microparticles, deep into colonic tissue ex vivo. Delivery was relatively independent of size and charge but did depend on conformation, with regular, spherical particles being delivered to a greater extent than long-chain polymers. The subsequent residence time of model permeants in tissue after ultrasound-mediated delivery was found to depend on size, with large microparticles demonstrating negligible clearance from the local tissue 24h after delivery ex vivo. The dependence of clearance time on permeant size was further confirmed in vivo in mice using fluorescently labeled 3kDa and 70kDa dextran. The use of low-frequency ultrasound in the GI tract represents a novel tool for the delivery of a wide-range of therapeutics independent of formulation, potentially allowing for the tailoring of formulations to impart novel pharmacokinetic profiles once delivered into tissue.

Novel protease inhibitor-loaded Nanoparticle-in-Microparticle Delivery System leads to a dramatic improvement of the oral pharmacokinetics in dogs.

With the advent of the Highly Active Antiretroviral Therapy, the morbidity and the mortality associated to HIV have been considerably reduced. However, 35-40 million people bear the infection worldwide. One of the main causes of therapeutic failure is the frequent administration of several antiretrovirals that results in low patient compliance and treatment cessation. In this work, we have developed an innovative Nanoparticle-in-Microparticle Delivery System (NiMDS) comprised of pure drug nanocrystals of the potent protease inhibitor indinavir free base (used as poorly water-soluble model protease inhibitor) produced by nanoprecipitation that were encapsulated within mucoadhesive polymeric microparticles. Pure drug nanoparticles and microparticles were thoroughly characterized by diverse complementary techniques. NiMDSs displayed an encapsulation efficiency of approximately 100% and a drug loading capacity of up to 43% w/w. In addition, mucoadhesiveness assays ex vivo conducted with bovine gut showed that film-coated microparticles were retained for more than 6 h. Finally, pharmacokinetics studies in mongrel dogs showed a dramatic 47- and 95-fold increase of the drug oral bioavailability and half-life, respectively, with respect to the free unprocessed drug. These results support the outstanding performance of this platform to reduce the dose and the frequency of administration of protease inhibitors, a crucial step to overcome the current patient-incompliant therapy.