Enzyme-Triggered Adhesives In article 2301033, Giovanni Traverso and co-workers develop an oral sandwiched pill with in situ gelling systems gelled based on enzyme catalyzed crosslinking reactions, which undergo mild conditions, tunable gelation rate, and tough mechanical properties by a simple mixing to deliver clinically-relevant doses of hydrophilic and hydrophobic drugs in a large animal model, with a sustained half life and Tmax compared to conventional pills. This work is seen as a transformative step in drug delivery research that ushers in a new era of oral sustained drug delivery: The invention can not only be used to orally deliver drugs to optimize drugs pharmacokinetics after oral administration, but also can be used in gastrointestinal (GI) diagnoses application to ensure the diagnostic device stays in the GI tract for enough time.
Epithelial tissues line the organs of the body, providing an initial protective barrier as well as a surface for nutrient and drug absorption. Here, we identified enzymatic components present in the gastrointestinal epithelium that can serve as selective means for tissue-directed polymerization. We focused on the small intestine, given its role in drug and nutrient absorption and identified catalase as an essential enzyme with the potential to catalyze polymerization and growth of synthetic biomaterial layers. We demonstrated that the polymerization of dopamine by catalase yields strong tissue adhesion. We characterized the mechanism and specificity of the polymerization in segments of the gastrointestinal tracts of pigs and humans ex vivo. Moreover, we demonstrated proof of concept for application of these gastrointestinal synthetic epithelial linings for drug delivery, enzymatic immobilization for digestive supplementation, and nutritional modulation through transient barrier formation in pigs. This catalase-based approach to in situ biomaterial generation may have broad indications for gastrointestinal applications.
Purpose: Poor wound healing outcomes are common in patients seeking gender-affirming surgery and our prior work implicates exogenous testosterone (T) as a contributor. However, cessation of testosterone is associated with impaired quality-of-life. Further, blockade of deleterious effects of endogenous T in cis-males at the wound site is desirable. Similarly, estradiol has been shown to exert pro-wound repair effects via estrogen receptor (ER). We sought to develop sustained-release topical therapeutics for the delivery of 17β-estradiol and flutamide (an ER agonist and AR antagonist respectively) to improve healing of cutaneous wounds. Methods: Three different formulations of topical 17b-estrogen and one of flutamide were created. Active agents were mixed at different concentrations with propylene glycol monocaprylate (cosurfactant, humectant, solubilizer), stearyl alcohol (emulsifier/gelling agent), water, and coconut oil (vehicle). Topicals underwent stability and in vitro permeation/release tests using rat skin in a Franz vertical diffusion cell. Ointment was applied to rat skin in the donor compartment. Samples were taken from the receptor compartment, containing PBS plus methyl-β-cyclodextrin (MβCD), at timepoints of 30 minutes, 1-8h, and 24h then diluted in methanol, centrifuged, and underwent high-performance liquid chromatography. Rat skin samples were homogenized, centrifuged and supernatant subsequently analyzed using HPLC. A flutamide and 17b-estradiol ointment were tested in our rat model of testosterone-influenced WH. Rats underwent surgical castration and were randomly allocated based on systemic hormone and topical therapy (no systemic hormone vs. systemic testosterone cypionate; topical 17b-Estradiol vs. Flutamide vs. vehicle applied daily). Animals underwent bilateral dorsal excisional wounding, were harvested on POD10, and analyzed with planimetry, histology, and immunofluorescence. Systemic testosterone and estradiol levels were measured with mass spectrometry. Results: Estradiol levels were detected in all skin samples treated with 17b-estradiol formulations (55.2ug formulation A, 25.8ug formulation B, 37.7ug formulation C), showing wound delivery. Estradiol levels were undetectable on all nine Franz diffusion cell runs across all formulations, suggesting negligible systemic release. Wounds from rats with exogenous T and topically treated with estradiol (OVX/E2 + 2T) or Flutamide (OVX/FLUT+2T) experienced faster wound closure rates at days 3 and 5 compared to OVX/2T (p=0.0019 and p=0.0069 respectively). Histology revealed increased granulation thickness in OVX/E2 + 2T and OVX/FLUT+2T rats compared to controls. Wounds from OVX/E2 + 2T had decreased epithelial gap and increased epithelial thickness compared to OVX/FLUT+2T (p<0.05). Immunofluorescence staining revealed higher expression of CD68 in OVX/FLUT+2T and OVX/E2 + 2T compared to OVX/PLAC (p=0.0077 and p=0.0461 respectively). ER biomarkers showed higher expression in the OVX/E2 + 2T group as compared to OVX/PLAC. Conclusion: We developed proof-of-concept 17b-estradiol and flutamide sustained-release topical formulations to improve wound repair. Stability, permeation tests, and E2 levels on the skin and transdermal compartment revealed all three topical estradiol formulations were stable and permeated into the skin adequately with minimal transdermal penetration, suggesting these formulations may offer local delivery with minimal systemic delivery. Ongoing efforts include improvements in formulation (e.g., addition of stabilizers) and conduction of further animal efficacy experiments.
Abstract The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic subtypes of malignant glioma are sensitive to selective inhibition of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT). However, the potential impact of NAD+ depletion on the brain tumor microenvironment has not been elaborated. In addition, systemic toxicity of NAMPT inhibition remains a significant concern. Here we show that microparticle-mediated intratumoral delivery of NAMPT inhibitor GMX1778 induces specific immunologic changes in the tumor microenvironment of murine GBM, characterized by upregulation of immune checkpoint PD-L1, recruitment of CD3+, CD4+, and CD8+ T cells, and reduction of M2-polarized immunosuppressive macrophages. NAD+ depletion and autophagy induced by NAMPT inhibitors mediated the upregulation of PD-L1 transcripts and cell surface protein levels in GBM cells. NAMPT inhibitor modulation of the tumor immune microenvironment was therefore combined with PD-1 checkpoint blockade in vivo, significantly increasing the survival of GBM-bearing animals. Thus, the therapeutic impacts of NAMPT inhibition extended beyond neoplastic cells, shaping surrounding immune effectors. Microparticle delivery and release of NAMPT inhibitor at the tumor site offers a safe and robust means to alter an immune tumor microenvironment that could potentiate checkpoint immunotherapy for glioblastoma. Significance: Microparticle-mediated local inhibition of NAMPT modulates the tumor immune microenvironment and acts cooperatively with anti-PD-1 checkpoint blockade, offering a combination immunotherapy strategy for the treatment of GBM.
<p>Supplemental figures 1-10. Supplementary Figure S1. Cell viability assay with NAMPT inhibitors. Supplementary Figure S2. NAMPT inhibitor induces PD-L1 upregulation on alive cells. Supplementary Figure S3. NAMPT inhibitor induces PD-L1 upregulation. Supplementary Figure S4. Tumor PD-L1 upregulation after injection of coumarin6GMX1778 co-loaded microparticles. Supplementary Figure S5. NAMPT inhibitor-induced autophagy underlies an increase in PD-L1 mRNA and protein levels. Supplementary Figure S6. Impact of local treatment with GMX1778 microparticles on cells labeled with Arg1 and CD68 in murine glioblastoma. Supplementary Figure S7. NAMPT inhibitor decreases glioblastoma-associated macrophages. Supplementary Figure S8. PD-L1 immunohistochemistry (brown) of GL261 glioblastoma after treatment with blank micro-particles (MP), blank MP and anti-PD-1, GMX1778 MP, and combination of GMX1778 MP and anti-PD-1. Supplementary Figure S9. Immunoflurescence of GL261 glioblastoma after treatment with blank micro-particles, anti-PD-1, GMX1778 micro-particles, and combination. Supplementary Figure S10. GranzymeB immunohistochemistry (brown) of GL261 glioblastoma after treatment with blank micro-particles (MP), blank MP and anti-PD-1, GMX1778 MP, and combination of GMX1778 MP and anti-PD-1.</p>
Abstract The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic sub-types of malignant glioma are sensitive to selective inhibition of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT). However, the potential impact of NAD+ depletion on the brain tumor microenvironment has not been elaborated. In addition, systemic toxicity of NAMPT inhibition remains a significant concern. Here, we show that microparticle-mediated intratumoral delivery of NAMPT inhibitor GMX1778 induces specific immunological changes in the tumor microenvironment of murine GBM, characterized by upregulation of immune checkpoint PD-L1, recruitment of CD3+, CD4+ and CD8+ T cells and reduction of M2-polarized immunosuppressive macrophages. NAD+ depletion and autophagy induced by NAMPT inhibitors mediated the upregulation of PD-L1 transcripts and cell surface protein levels in GBM cells. NAMPT inhibitor modulation of the tumor immune microenvironment was therefore combined with PD-1 checkpoint blockade in vivo, significantly increasing the survival of GBM bearing animals. Thus, the therapeutic impacts of NAMPT inhibition extended beyond neoplastic cells, shaping surrounding immune effectors. Microparticle delivery and release of NAMPT inhibitor at the tumor site offers a safe and robust means to alter an immune tumor microenvironment that could potentiate checkpoint immunotherapy for glioblastoma.
MicroRNAs are small (18-22 nucleotide long) noncoding RNAs that play important roles in biological processes through posttranscriptional regulation of gene expression. Their aberrant expression and functional significance are reported in several human malignancies, including pancreatic cancer. Recently, we identified miR-150 as a novel tumor suppressor microRNA in pancreatic cancer. Furthermore, expression of miR-150 was downregulated in the majority of tumor cases, suggesting that its restoration could serve as an effective approach for pancreatic cancer therapy. In the present study, we developed a nanoparticle-based miR-150 delivery system and tested its therapeutic efficacy in vitro. Using double emulsion solvent evaporation method, we developed a poly (D,L-lactide-co-glycolide) (PLGA)-based nanoformulation of miR-150 (miR-150-NF). Polyethyleneimine (a cationic polymer) was incorporated in PLGA matrix to increase the encapsulation of miR-150. Physical characterization of miR-150-NF demonstrated that these nanoparticles had high encapsulation efficiency (~78%) and exhibited sustained release profile. Treatment of pancreatic cancer cells with miR-150-NF led to efficient intracellular delivery of miR-150 mimics and caused significant downregulation of its target gene (MUC4) expression. Inhibition of MUC4 correlated with a concomitant decrease in the expression of its interacting partner, HER2, and repression of its downstream signaling. Furthermore, treatment of pancreatic cancer cells with miR-150-NF suppressed their growth, clonogenicity, motility, and invasion. Together, these findings suggest that PLGA-based nanoformulation could potentially serve as a safe and effective nanovector platform for miR-150 delivery to pancreatic tumor cells.
The development of oral sustained release dosage forms has been a longstanding goal due to the potential for ease of administration, improved pharmacokinetics, reduced dosing frequency, and improved adherence. The benefits of multiday single-dose drug delivery are evident in the success and patient adoption of injected and implanted dosage forms. However, in the space of oral medications, all current commercially available gastric resident dosage forms, and most in development, are limited to gastric residence of less than 1 day.Reviews of systems to extend gastric residence reveal that 1 day or more residence has been an unmet challenge. New dosage forms are in development that seek to address many of the key physiological and design challenges of long-term gastric retention beyond 24 h and up to a week or longer. The present analysis highlights the design, material considerations and implications of unfolding dosage form systems with ultra-long-term gastric residence.The development of oral dosage forms providing sustained release of high potency medications over days or weeks could transform care, significantly decrease patient burden in chronic disease management and improve outcomes.
