Multidimensional opioid abuse deterrence using a nanoparticle-polymer hybrid formulation.

Misuse of prescription opioid drugs is the leading cause of the opioid crisis and overdose-related death. Abuse deterrent formulations (ADFs) have been developed to discourage attempts to tamper with the formulation and alter the ingestion methods. However, abusers develop complex extraction strategies to circumvent the ADF technologies. For comprehensive deterrence of drug abuse, we develop tannic acid nanoparticles (NPs) that protect encapsulated opioids from solvent extraction and thermal challenge (crisping), complementing the existing formulation strategy to deter injection abuse. Here, we develop a hybrid ADF tablet (NP-Tab), consisting of iron-crosslinked tannic acid NPs encapsulating thebaine (model opioid compound), xanthan gum, and chitosan (gel-forming polymers), and evaluate its performance in common abuse conditions. NP-Tab tampered by crushing and suspended in aqueous solvents forms an instantaneous gel, which is difficult to pull or push through a 21-gauge needle. NPs insulate the drug from organic solvents, deterring solvent extraction. NPs also promote thermal destruction of the drug to make crisping less rewarding. However, NP-Tab releases thebaine in the simulated gastric fluid without delay, suggesting that its analgesic effect may be unaffected if consumed orally as prescribed. These results demonstrate that NP-Tab can provide comprehensive drug abuse deterrence, resisting aqueous/organic solvent extraction, injection, and crisping, while retaining its therapeutic effect upon regular usage.

Delivery of STING agonists for cancer immunotherapy.

Agonists of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway, a critical mediator of innate immune response to foreign invaders with DNA, have gained significant interest in cancer immunotherapy. STING agonists are envisioned as a way of complementing the antitumor activity of the patient's immune system and immune checkpoint blockade therapy. However, their clinical development has been challenging due to the poor pharmacokinetic and physicochemical properties. This review discusses drug delivery efforts to circumvent the challenges, their accomplishment, and unmet needs based on the last five years of literature.

Recent trends in the delivery of RNA drugs: Beyond the liver, more than vaccine.

RNAs are known for versatile functions and therapeutic utility. They have gained significant interest since the approval of several RNA drugs, including COVID-19 mRNA vaccines and therapeutic agents targeting liver diseases. There are increasing expectations for a new class of RNA drugs for broader applications. Successful development of RNA drugs for new applications hinges on understanding their diverse functions and structures. In this review, we explore the last five years of literature to understand current approaches to formulate a spectrum of RNA drugs, focusing on new efforts to expand their applications beyond vaccines and liver diseases.

Systemic Delivery of Paclitaxel by Find-Me Nanoparticles Activates Antitumor Immunity and Eliminates Tumors.

Local delivery of immune-activating agents has shown promise in overcoming an immunosuppressive tumor microenvironment (TME) and stimulating antitumor immune responses in tumors. However, systemic therapy is ultimately needed to treat tumors that are not readily locatable or accessible. To enable systemic delivery of immune-activating agents, we employ poly(lactic-co-glycolide) (PLGA) nanoparticles (NPs) with a track record in systemic application. The surface of PLGA NPs is decorated with adenosine triphosphate (ATP), a damage-associated molecular pattern to recruit antigen-presenting cells (APCs). The ATP-conjugated PLGA NPs (NPpD-ATP) are loaded with paclitaxel (PTX), a chemotherapeutic agent inducing immunogenic cell death to generate tumor antigens in situ. We show that the NPpD-ATP retains ATP activity in hostile TME and provides a stable "find-me" signal to recruit APCs. Therefore, the PTX-loaded NPpD-ATP helps populate antitumor immune cells in TME and attenuate the growth of CT26 and B16F10 tumors better than a mixture of PTX-loaded NPpD and ATP. Combined with anti-PD-1 antibody, PTX-loaded NPpD-ATP achieves complete regression of CT26 tumors followed by antitumor immune memory. This study demonstrates the feasibility of systemic immunotherapy using a PLGA NP formulation that delivers ICD-inducing chemotherapy and an immunostimulatory signal.

Long-Acting Microparticle Formulation of Griseofulvin for Ocular Neovascularization Therapy.

Neovascular age-related macular degeneration (nAMD) is a leading cause of vision loss in older adults. nAMD is treated with biologics targeting vascular endothelial growth factor; however, many patients do not respond to the current therapy. Here, a small molecule drug, griseofulvin (GRF), is used due to its inhibitory effect on ferrochelatase, an enzyme important for choroidal neovascularization (CNV). For local and sustained delivery to the eyes, GRF is encapsulated in microparticles based on poly(lactide-co-glycolide) (PLGA), a biodegradable polymer with a track record in long-acting formulations. The GRF-loaded PLGA microparticles (GRF MPs) are designed for intravitreal application, considering constraints in size, drug loading content, and drug release kinetics. Magnesium hydroxide is co-encapsulated to enable sustained GRF release over >30 days in phosphate-buffered saline with Tween 80. Incubated in cell culture medium over 30 days, the GRF MPs and the released drug show antiangiogenic effects in retinal endothelial cells. A single intravitreal injection of MPs containing 0.18 µg GRF releases the drug over 6 weeks in vivo to inhibit the progression of laser-induced CNV in mice with no abnormality in the fundus and retina. Intravitreally administered GRF MPs prove effective in preventing CNV, providing proof-of-concept toward a novel, cost-effective nAMD therapy.

RNA helicase DDX5 modulates sorafenib sensitivity in hepatocellular carcinoma via the Wnt/ß-catenin-ferroptosis axis.

Reduced expression of the RNA helicase DDX5 associated with increased hepatocellular carcinoma (HCC) tumor grade and poor patient survival following treatment with sorafenib. While immunotherapy is the first-line treatment for HCC, sorafenib and other multi-tyrosine kinase inhibitors (mTKIs) are widely used when immunotherapy is contra-indicated or fails. Herein, we elucidate the role of DDX5 in sensitizing HCC to sorafenib, offering new therapeutic strategies. Treatment of various human HCC cell lines with sorafenib/mTKIs downregulated DDX5 in vitro and in preclinical HCC models. Conversely, DDX5 overexpression reduced the viability of sorafenib-treated cells via ferroptosis, suggesting a role for DDX5 in sorafenib sensitivity. RNAseq of wild-type vs. DDX5-knockdown cells treated with or without sorafenib identified a set of common genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps with Wnt signaling genes, including Disheveled-1 (DVL1), an indispensable Wnt activator and prognostic indicator of poor survival for sorafenib-treated patients. DDX5-knockout (DDX5KO) HCC cells exhibited DVL1 induction, Wnt/ß-catenin pathway activation, and ferroptosis upon inhibition of canonical Wnt signaling. Consistently, xenograft HCC tumors exhibited reduced growth by inhibition of Wnt/ß-catenin signaling via induction of ferroptosis. Significantly, overexpression of DDX5 in HCC xenografts repressed DVL1 expression and increased ferroptosis, resulting in reduced tumor growth by sorafenib. We conclude that DDX5 downregulation by sorafenib mediates adaptive resistance by activating Wnt/ß-catenin signaling, leading to ferroptosis escape. Conversely, overexpression of DDX5 in vivo enhances the anti-tumor efficacy of sorafenib by suppressing Wnt/ß-catenin activation and induction of ferroptosis. Thus, DDX5 overexpression in combination with mTKIs is a promising therapeutic strategy for HCC.

Interpenetrating Networks of Collagen and Hyaluronic Acid That Serve as In Vitro Tissue Models for Assessing Macromolecular Transport.

High-fidelity preclinical in vitro tissue models can reduce the failure rate of drugs entering clinical trials. Collagen and hyaluronic acid (HA) are major components of the extracellular matrix of many native tissues and affect therapeutic macromolecule diffusion and recovery through tissues. Although collagen and HA are commonly used in tissue engineering, the physical and mechanical properties of these materials are variable and depend highly on processing conditions. In this study, HA was chemically modified and crosslinked via hydrazone bonds to form interpenetrating networks of crosslinked HA (HAX) with collagen (Col). These networks enabled a wide range of mechanical properties, including stiffness and swellability, and microstructures, such as pore morphology and size, that can better recapitulate diverse tissues. We utilized these interpenetrating ColHAX hydrogels as in vitro tissue models to examine macromolecular transport and recovery for early-stage drug screening. Hydrogel formulations with varying collagen and HAX concentrations imparted different gel properties based on the ratio of collagen to HAX. These gels were stable and swelled up to 170% of their original mass, and the storage moduli of the ColHAX gels increased over an order of magnitude by increasing collagen and HA concentration. Interestingly, when HAX concentration was constant and collagen concentration increased, both the pore size and spatial colocalization of collagen and HA increased. HA in the system dominated the ζ-potentials of the gels. The hydrogel and macromolecule properties impacted the mass transport and recovery of lysozyme, ß-lactoglobulin, and bovine serum albumin (BSA) from the ColHAX gels─large molecules were largely impacted by mesh size, whereas small molecules were influenced primarily by electrostatic forces. Overall, the tunable properties demonstrated by the ColHAX hydrogels can be used to mimic different tissues for early-stage assays to understand drug transport and its relationship to matrix properties.

Control of drug release kinetics from hot-melt extruded drug-loaded polycaprolactone matrices.

Sustained local delivery of meloxicam by polymeric structures is desirable for preventing subacute inflammation and biofilm formation following tissue incision or injury. Our previous study demonstrated that meloxicam release from hot-melt extruded (HME) poly(ε-caprolactone) (PCL) matrices could be controlled by adjusting the drug content. Increasing drug content accelerated the drug release as the initial drug release generated a pore network to facilitate subsequent drug dissolution and diffusion. In this study, high-resolution micro-computed tomography (HR µCT) and artificial intelligence (AI) image analysis were used to visualize the microstructure of matrices and simulate the drug release process. The image analysis indicated that meloxicam release from the PCL matrix was primarily driven by diffusion but limited by the amount of infiltrating fluid when drug content was low (i.e., the connectivity of the drug/pore network was poor). Since the drug content is not easy to change when a product has a fixed dose and dimension/geometry, we sought an alternative approach to control the meloxicam release from the PCL matrices. Here, magnesium hydroxide (Mg(OH)2) was employed as a solid porogen in the drug-PCL matrix so that Mg(OH)2 dissolved with time in the aqueous environment creating additional pore networks to facilitate local dissolution and diffusion of meloxicam. PCL matrices were produced with a fixed 30 wt% meloxicam loading and variable Mg(OH)2 loadings from 20 wt% to 50 wt%. The meloxicam release increased in proportion to the Mg(OH)2 content, resulting in almost complete drug release in 14 d from the matrix with 50 wt% Mg(OH)2. The porogen addition is a simple strategy to tune drug release kinetics, applicable to other drug-eluting matrices with similar constraints.

A timescale-guided microfluidic synthesis of tannic acid-FeIII network nanocapsules of hydrophobic drugs.

Many drugs are poorly water-soluble and suffer from low bioavailability. Metal-phenolic network (MPN), a hydrophilic thin layer such as tannic acid (TA)-FeIII network, has been recently used to encapsulate hydrophobic drugs to improve their bioavailability. However, it remains challenging to synthesize nanocapsules of a wide variety of hydrophobic drugs and to scale up the production in a continuous manner. Here, we present a microfluidic synthesis method to continuously produce TA-FeIII network nanocapsules of hydrophobic drugs. We hypothesize that nanocapsules can continuously be formed only when the microfluidic mixing timescale is shorter than the drug's nucleation timescale. The hypothesis was tested on three hydrophobic drugs - paclitaxel, curcumin, and vitamin D with varying solubility and nucleation timescale. The proposed mechanism was validated by successfully predicting the synthesis outcomes. The microfluidically-synthesized nanocapsules had well-controlled sizes of 100-200 nm, high drug loadings of 40-70%, and a throughput of up to 70 mg hr-1 per channel. The release kinetics, cellular uptake, and cytotoxicity were further evaluated. The effect of coating constituents on nanocapsule properties were characterized. Fe content of nanocapsules was reported. The stability of nanocapsules at different temperatures and pHs were also tested. The results suggest that the present method can provide a quantitative guideline to predictively design a continuous synthesis scheme for hydrophobic drug encapsulation via MPN nanocapsules with scaled-up capability.

Susceptibility to SARS-CoV-2 and MERS-CoV in Beagle Dogs.

The coronavirus disease 19 (COVID-19) pandemic, caused by the severe acute respiratory syndrome, coronavirus 2 (SARS-CoV-2), has resulted in unprecedented challenges to healthcare worldwide. In particular, the anthroponotic transmission of human coronaviruses has become a common concern among pet owners. Here, we experimentally inoculated beagle dogs with SARS-CoV-2 or Middle East respiratory syndrome (MERS-CoV) to compare their susceptibility to and the pathogenicity of these viruses. The dogs in this study exhibited weight loss and increased body temperatures and shed the viruses in their nasal secretions, feces, and urine. Pathologic changes were observed in the lungs of the dogs inoculated with SARS-CoV-2 or MERS-CoV. Additionally, clinical characteristics of SARS-CoV-2, such as increased lactate dehydrogenase levels, were identified in the current study.

Protein corona: Friend or foe? Co-opting serum proteins for nanoparticle delivery.

For systemically delivered nanoparticles to reach target tissues, they must first circulate long enough to reach the target and extravasate there. A challenge is that the particles end up engaging with serum proteins and undergo immune cell recognition and premature clearance. The serum protein binding, also known as protein corona formation, is difficult to prevent, even with artificial protection via "stealth" coating. Protein corona may be problematic as it can interfere with the interaction of targeting ligands with tissue-specific receptors and abrogate the so-called active targeting process, hence, the efficiency of drug delivery. However, recent studies show that serum protein binding to circulating nanoparticles may be actively exploited to enhance their downstream delivery. This review summarizes known issues of protein corona and traditional strategies to control the corona, such as avoiding or overriding its formation, as well as emerging efforts to enhance drug delivery to target organs via nanoparticles. It concludes with a discussion of prevailing challenges in exploiting protein corona for nanoparticle development.

Development of poly(lactide-co-glycolide) microparticles for sustained delivery of meloxicam.

Poly(lactide-co-glycolide) (PLGA) polymers have been widely used for drug delivery due to their biodegradability and biocompatibility. One of the objectives of encapsulating a drug in PLGA microparticles (MPs) is to achieve an extended supply of the drug through sustained release, which can range from weeks to months. Focusing on the applications needing a relatively short-term delivery, we investigated formulation strategies to achieve a drug release from PLGA MPs for two weeks, using meloxicam as a model compound. PLGA MPs produced by the traditional oil/water (O/W) single emulsion method showed only an initial burst release with minimal increase in later-phase drug release. Alternatively, encapsulating meloxicam as solid helped reduce the initial burst release. The inclusion of magnesium hydroxide [Mg(OH)2] enhanced later-phase drug release by neutralizing the developing acidity that limited the drug dissolution. The variation of solid meloxicam and Mg(OH)2 quantities allowed for flexible control of meloxicam release, yielding MPs with distinct in vitro release kinetics. When subcutaneously injected into rats, the MPs with relatively slow in vitro drug release kinetics showed in vivo drug absorption profiles consistent with in vitro trend. However, the MPs that rapidly released meloxicam showed an attenuated in vivo absorption, suggesting premature precipitation of fast-released meloxicam. In summary, this study demonstrated the feasibility of controlling drug release from the PLGA MPs over weeks based on the physical state of the encapsulated drug and the inclusion of Mg(OH)2 to neutralize the microenvironmental pH of the MPs.

Size optimization of carfilzomib nanocrystals for systemic delivery to solid tumors.

Carfilzomib (CFZ) is a second-generation proteasome inhibitor effective in blood cancer therapy. However, CFZ has shown limited efficacy in solid tumor therapy due to the short half-life and poor tumor distribution. Albumin-coated nanocrystal (NC) formulation was shown to improve the circulation stability of CFZ, but its antitumor efficacy remained suboptimal. We hypothesize that NC size reduction is critical to the formulation safety and efficacy as the small size would decrease the distribution in the reticuloendothelial system (RES) and selectively increase the uptake by tumor cells. We controlled the size of CFZ-NCs by varying the production parameters in the crystallization-in-medium method and compared the size-reduced CFZ-NCs (z-average of 168 nm, NC168) with a larger counterpart (z-average of 325 nm, NC325) as well as the commercial CFZ formulation (CFZ-CD). Both CFZ-NCs showed similar or higher cytotoxicity than CFZ-CD against breast cancer cells. NC168 showed greater uptake by cancer cells, less uptake by macrophages and lower immune cell toxicity than NC325 or CFZ-CD. NC168, but not NC325, showed a similar safety profile to CFZ-CD in vivo. The biodistribution and antitumor efficacy of CFZ-NCs in mice were also size-dependent. NC168 showed greater antitumor efficacy and tumor accumulation but lower RES accumulation than NC325 in 4T1 breast cancer model. These results support that NC formulation with an optimal particle size can improve the therapeutic efficacy of CFZ in solid tumors.

PLA-PCL microsphere formulation to deter abuse of prescription opioids by smoking.

Opioids are commonly prescribed across the United States (US) for pain relief, despite their highly addictive nature that often leads to abuse and overdose deaths. Abuse deterrent formulations (ADFs) for prescription opioids make the non-therapeutic use of these drugs more difficult and less satisfying. Although approximately one-third of surveyed abusers in the US reported smoking opioids, to our knowledge, no commercialized ADF effectively prevents opioid smoking. Here, we report a novel approach to deter smoking of a model prescription opioid drug, thebaine (THB), by using polymer blend microspheres (MS) comprising polylactic acid (PLA) and polycaprolactone (PCL). We utilized high-performance liquid chromatography (HPLC) and thermogravimetric analysis (TGA) to test the ability of PLA-PCL MS to limit the escape of vaporized THB. Additionally, we compared the abuse-deterrent potential of PLA-PCL MS to that of activated carbon (AC) and mesoporous silica (MPS), two materials with excellent drug-adsorbing properties. Our MS formulation was effective in reducing the amount of both active drug and thermal degradation products in the vapor generated upon heating of THB. These results support that PLA-PCL microspheres can be co-formulated in a tablet with common prescription opioids to deter their abuse via the smoking route.

Severe acute respiratory syndrome coronavirus 2 and influenza A virus co-infection alters viral tropism and haematological composition in Syrian hamsters.

The ongoing coronavirus disease 2019 pandemic and its overlap with the influenza season lead to concerns over severe disease caused by the influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) co-infections. Using a Syrian hamster co-infection model with SARS-CoV-2 and the pandemic influenza virus A/California/04/2009 (H1N1), we found (a) more severe disease in co-infected animals, compared to those infected with influenza virus alone but not SARS-CoV-2 infection alone; (b) altered haematological changes in only co-infected animals and (c) altered influenza virus tropism in the respiratory tracts of co-infected animals. Overall, our study revealed that co-infection with SARS-CoV-2 and influenza virus is associated with altered disease severity and tissue tropism, as well as haematological changes, compared to infection with either virus alone.

Conditional Convolution Projecting Latent Vectors on Condition-Specific Space.

Despite rapid advancements over the past several years, the conditional generative adversarial networks (cGANs) are still far from being perfect. Although one of the major concerns of the cGANs is how to provide the conditional information to the generator, there are not only no ways considered as the optimal solution but also a lack of related research. This brief presents a novel convolution layer, called the conditional convolution (cConv) layer, which incorporates the conditional information into the generator of the generative adversarial networks (GANs). Unlike the most general framework of the cGANs using the conditional batch normalization (cBN) that transforms the normalized feature maps after convolution, the proposed method directly produces conditional features by adjusting the convolutional kernels depending on the conditions. More specifically, in each cConv layer, the weights are conditioned in a simple but effective way through filter-wise scaling and channel-wise shifting operations. In contrast to the conventional methods, the proposed method with a single generator can effectively handle condition-specific characteristics. The experimental results on CIFAR, LSUN, and ImageNet datasets show that the generator with the proposed cConv layer achieves a higher quality of conditional image generation than that with the standard convolution layer.

A single local delivery of paclitaxel and nucleic acids via an immunoactive polymer eliminates tumors and induces antitumor immunity.

Despite recent advances in cancer therapy, hard-to-reach, unidentified tumors remain a significant clinical challenge. A promising approach is to treat locatable and accessible tumors locally and stimulate antitumor immunity in situ to exert systemic effects against distant tumors. We hypothesize that a carrier of immunotherapeutics can play a critical role in activating antitumor immunity as an immunoadjuvant and a local retainer of drug combinations. Here, we develop a polyethyleneimine-lithocholic acid conjugate (2E'), which forms a hydrophobic core and cationic surface to codeliver hydrophobic small molecules and anionic nucleic acids and activates antigen-presenting cells via the intrinsic activities of 2E' components. 2E' delivers paclitaxel and small-interfering RNA (siRNA) targeting PD-L1 (or cyclic dinucleotide, [CDN]) to induce the immunogenic death of tumor cells and maintain the immunoactive tumor microenvironment, and further activates dendritic cells and macrophages, leveraging the activities of loaded drugs. A single local administration of 2E' or its combination with paclitaxel and PD-L1­targeting siRNA or CDN induces strong antitumor immunity, resulting in immediate regression of large established tumors, tumor-free survival, an abscopal effect on distant tumors, and resistance to rechallenge and metastasis in multiple models of murine tumors, including CT26 colon carcinoma, B16F10 melanoma, and 4T1 breast cancer. This study supports the finding that local administration of immunotherapeutics, when accompanied by the rationally designed carrier, can effectively protect the host from distant and recurrent diseases.

Nucleic acid and oligonucleotide delivery for activating innate immunity in cancer immunotherapy.

A group of nucleic acids and oligonucleotides play various roles in the innate immune system. They can stimulate pattern recognition receptors to activate innate immune cells, encode immunostimulatory proteins or peptides, or silence specific genes to block negative regulators of immune cells. Given the limitations of current cancer immunotherapy, there has been increasing interest in harnessing innate immune responses by nucleic acids and oligonucleotides. The poor biopharmaceutical properties of nucleic acids and oligonucleotides make it critical to use carriers that can protect them in circulation, retain them in the tumor microenvironment, and bring them to intracellular targets. Therefore, various gene carriers have been repurposed to deliver nucleic acids and oligonucleotides for cancer immunotherapy and improve their safety and activity. Here, we review recent studies that employed carriers to enhance the functions of nucleic acids and oligonucleotides and overall immune responses to cancer, and discuss remaining challenges and future opportunities in the development of nucleic acid-based immunotherapeutics.