Qingguo Xu

A Dense Poly(Ethylene Glycol) Coating Improves Penetration of Large Polymeric Nanoparticles Within Brain Tissue

Nanoparticles densely coated with poly(ethylene glycol) rapidly penetrate within mouse, rat, and human brain parenchyma. Brain-Penetrating Particles It was once thought that particles larger than 60 nm would be stuck in the brain extracellular space (ECS), unable to penetrate further. This has been a particularly bothersome rule of thumb for the design of drug delivery systems that rely on larger particles or viruses to carry therapeutics. Now, Nance and colleagues have challenged this hypothesis by exploring particles that are >60 nm, discovering that large particles, with the right coating, can indeed diffuse throughout the ECS of both rat and human brains. The authors first coated fluorescent polystyrene particles with a dense layer of the bio-inert polymer poly(ethylene glycol) (commonly known as PEG) or with a carboxyl moiety (COOH). Using a multiple-particle tracking method, the authors reported that 114-nm PEG-coated particles penetrated ex vivo human brain tissue with ease, whereas similarly sized COOH-coated particles were stopped in their tracks. Nance et al. attributed this difference to the dense, near-neutral PEG coating, claiming that it allows the particles to experience the brain ECS more as a fluid than an impermeable solid. The importance of the PEG coating was further confirmed in living mice, where real-time video microscopy revealed that the 100-nm PEG-coated particles penetrated farther into the mouse brain than the 100-nm COOH-coated ones. With a brain ECS pore size cutoff >100 nm, many doors can be opened in nanomedicine. Larger particles permit the inclusion of higher quantities of drug, which can be distributed for longer periods of time to more areas within the brain. Nance and colleagues preliminarily demonstrated such drug delivery capabilities using paclitaxel-loaded, 85-nm biodegradable nanoparticles, showing that only particles with the PEG coating could diffuse rapidly throughout rat brain tissue ex vivo. Although these densely coated particles may make drug delivery more efficient, they have yet to be tested in a disease model to confirm efficacy over conventional nanoparticles. Although currently limited to direct infusion into the brain, for eventual use in humans, it is hoped that they may be administered systemically for treating diseases with an impaired blood-brain barrier. Prevailing opinion suggests that only substances up to 64 nm in diameter can move at appreciable rates through the brain extracellular space (ECS). This size range is large enough to allow diffusion of signaling molecules, nutrients, and metabolic waste products, but too small to allow efficient penetration of most particulate drug delivery systems and viruses carrying therapeutic genes, thereby limiting effectiveness of many potential therapies. We analyzed the movements of nanoparticles of various diameters and surface coatings within fresh human and rat brain tissue ex vivo and mouse brain in vivo. Nanoparticles as large as 114 nm in diameter diffused within the human and rat brain, but only if they were densely coated with poly(ethylene glycol) (PEG). Using these minimally adhesive PEG-coated particles, we estimated that human brain tissue ECS has some pores larger than 200 nm and that more than one-quarter of all pores are ≥100 nm. These findings were confirmed in vivo in mice, where 40- and 100-nm, but not 200-nm, nanoparticles spread rapidly within brain tissue, only if densely coated with PEG. Similar results were observed in rat brain tissue with paclitaxel-loaded biodegradable nanoparticles of similar size (85 nm) and surface properties. The ability to achieve brain penetration with larger nanoparticles is expected to allow more uniform, longer-lasting, and effective delivery of drugs within the brain, and may find use in the treatment of brain tumors, stroke, neuroinflammation, and other brain diseases where the blood-brain barrier is compromised or where local delivery strategies are feasible.

Nanoparticle diffusion in, and microrheology of, the bovine vitreous ex vivo.

Intravitreal injection of biodegradable nanoparticles (NP) holds promise for gene therapy and drug delivery to the back of the eye. In some cases, including gene therapy, NP need to diffuse rapidly from the site of injection in order to reach targeted cell types in the back of the eye, whereas in other cases it may be preferred for the particles to remain at the injection site and slowly release drugs that may then diffuse to the site of action. We studied the movements of polystyrene (PS) NP of various sizes and surface chemistries in fresh bovine vitreous. PS NP as large as 510nm rapidly penetrated the vitreous gel when coated with polyethylene glycol (PEG), whereas the movements of NP 1190nm in diameter or larger were highly restricted regardless of surface chemistry owing to steric obstruction. PS NP coated with primary amine groups (NH2) possessed positively charged surfaces at the pH of bovine vitreous (pH=7.2), and were immobilized within the vitreous gel. In comparison, PS NP coated with COOH (possessing negatively charged surfaces) in the size range of 100-200nm and at particle concentrations below 0.0025% (w/v) readily diffused through the vitreous meshwork; at higher concentrations (~0.1% w/v), these nanoparticles aggregated within vitreous. Based on the mobility of different sized PEGylated PS NP (PS-PEG), we estimated the average mesh size of fresh bovine vitreous to be ~550±50nm. The bovine vitreous behaved as an impermeable elastic barrier to objects sized 1190nm and larger, but as a highly permeable viscoelastic liquid to non-adhesive objects smaller than 510nm in diameter. Guided by these studies, we next sought to examine the transport of drug- and DNA-loaded nanoparticles in bovine vitreous. Biodegradable NP with a diameter of 227nm, composed of a poly(lactic-co-glycolic acid) (PLGA)-based core coated with poly(vinyl alcohol) rapidly penetrated vitreous. Rod-shaped, highly-compacted CK30PEG10k/DNA with PEG coating (neutral surface charge; hydrodynamic diameter ~60nm) also diffused rapidly within vitreous. These findings will help guide the development of nanoparticle-based therapeutics for the treatment of vision-threatening ocular diseases.

Brain-penetrating nanoparticles improve paclitaxel efficacy in malignant glioma following local administration

Poor drug distribution and short drug half-life within tumors strongly limit efficacy of chemotherapies in most cancers, including primary brain tumors. Local or targeted drug delivery via controlled-release polymers is a promising strategy to treat infiltrative brain tumors, which cannot be completely removed surgically. However, drug penetration is limited with conventional local therapies since small-molecule drugs often enter the first cell they encounter and travel only short distances from the site of administration. Nanoparticles that avoid adhesive interactions with the tumor extracellular matrix may improve drug distribution and sustain drug release when applied to the tumor area. We have previously shown model polystyrene nanoparticles up to 114 nm in diameter were able to rapidly diffuse in normal brain tissue, but only if coated with an exceptionally dense layer of poly(ethylene glycol) (PEG) to reduce adhesive interactions. Here, we demonstrate that paclitaxel (PTX)-loaded, poly(lactic-co-glycolic acid) (PLGA)-co-PEG block copolymer nanoparticles with an average diameter of 70 nm were able to diffuse 100-fold faster than similarly sized PTX-loaded PLGA particles (without PEG coatings). Densely PEGylated PTX-loaded nanoparticles significantly delayed tumor growth following local administration to established brain tumors, as compared to PTX-loaded PLGA nanoparticles or unencapsulated PTX. Delayed tumor growth combined with enhanced distribution of drug-loaded PLGA-PEG nanoparticles to the tumor infiltrative front demonstrates that particle penetration within the brain tumor parenchyma improves therapeutic efficacy. The use of drug-loaded brain-penetrating nanoparticles is a promising approach to achieve sustained and more uniform drug delivery to treat aggressive gliomas and potentially other brain disorders.

Scalable method to produce biodegradable nanoparticles that rapidly penetrate human mucus.

Mucus typically traps and rapidly removes foreign particles from the airways, gastrointestinal tract, nasopharynx, female reproductive tract and the surface of the eye. Nanoparticles capable of rapid penetration through mucus can potentially avoid rapid clearance, and open significant opportunities for controlled drug delivery at mucosal surfaces. Here, we report an industrially scalable emulsification method to produce biodegradable mucus-penetrating particles (MPP). The emulsification of diblock copolymers of poly(lactic-co-glycolic acid) and polyethylene glycol (PLGA-PEG) using low molecular weight (MW) emulsifiers forms dense brush PEG coatings on nanoparticles that allow rapid nanoparticle penetration through fresh undiluted human mucus. In comparison, conventional high MW emulsifiers, such as polyvinyl alcohol (PVA), interrupts the PEG coating on nanoparticles, resulting in their immobilization in mucus owing to adhesive interactions with mucus mesh elements. PLGA-PEG nanoparticles with a wide range of PEG MW (1, 2, 5, and 10 kDa), prepared by the emulsification method using low MW emulsifiers, all rapidly penetrated mucus. A range of drugs, from hydrophobic small molecules to hydrophilic large biologics, can be efficiently loaded into biodegradable MPP using the method described. This readily scalable method should facilitate the production of MPP products for mucosal drug delivery, as well as potentially longer-circulating particles following intravenous administration.

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer.

Significance There are no effective therapies currently available for advanced pancreatic cancer. We show that there are two populations of cancer cells within a pancreatic tumor that require targeting by different metabolic inhibitors for effective tumor control. Rapidly dividing cells use glutamine, and can be effectively killed by administration of a nanoparticle containing an inhibitor of glutamine metabolism. Hypoxic cells, which are slowly dividing cells, metabolize glucose and can be targeted by metformin, a drug used for the treatment of diabetes. Clinical trials are needed to determine whether combination therapy, with drugs that effectively block the metabolism of glutamine and glucose, improves the survival of patients with pancreatic cancer. Targeting glutamine metabolism via pharmacological inhibition of glutaminase has been translated into clinical trials as a novel cancer therapy, but available drugs lack optimal safety and efficacy. In this study, we used a proprietary emulsification process to encapsulate bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a selective but relatively insoluble glutaminase inhibitor, in nanoparticles. BPTES nanoparticles demonstrated improved pharmacokinetics and efficacy compared with unencapsulated BPTES. In addition, BPTES nanoparticles had no effect on the plasma levels of liver enzymes in contrast to CB-839, a glutaminase inhibitor that is currently in clinical trials. In a mouse model using orthotopic transplantation of patient-derived pancreatic tumor tissue, BPTES nanoparticle monotherapy led to modest antitumor effects. Using the HypoxCR reporter in vivo, we found that glutaminase inhibition reduced tumor growth by specifically targeting proliferating cancer cells but did not affect hypoxic, noncycling cells. Metabolomics analyses revealed that surviving tumor cells following glutaminase inhibition were reliant on glycolysis and glycogen synthesis. Based on these findings, metformin was selected for combination therapy with BPTES nanoparticles, which resulted in significantly greater pancreatic tumor reduction than either treatment alone. Thus, targeting of multiple metabolic pathways, including effective inhibition of glutaminase by nanoparticle drug delivery, holds promise as a novel therapy for pancreatic cancer.

Corticosteroid-loaded biodegradable nanoparticles for prevention of corneal allograft rejection in rats

Immunologic graft rejection is one of the main causes of short and long-term graft failure in corneal transplantation. Steroids are the most commonly used immunosuppressive agents for postoperative management and prevention of corneal graft rejection. However, steroids delivered in eye drops are rapidly cleared from the surface of the eye, so the required frequency of dosing for corneal graft rejection management can be as high as once every 2h. Additionally, these eye drops are often prescribed for daily use for 1 year or longer, which can result in poor patient compliance and steroid-related side effects. Here, we report a biodegradable nanoparticle system composed of Generally Regarded as Safe (GRAS) materials that can provide sustained release of corticosteroids to prevent corneal graft rejection following subconjunctival injection provided initially during transplant surgery. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing dexamethasone sodium phosphate (DSP) exhibited a size of 200 nm, 8 wt.% drug loading, and sustained drug release over 15 days in vitro under sink conditions. DSP-loaded nanoparticles provided sustained ocular drug levels for at least 7 days after subconjunctival administration in rats, and prevented corneal allograft rejection over the entire 9-week study when administered weekly. In contrast, control treatment groups that received weekly injections of either placebo nanoparticles, saline, or DSP in solution demonstrated corneal graft rejection accompanied by severe corneal edema, neovascularization and opacity that occurred in ≤ 4 weeks. Local controlled release of corticosteroids may reduce the rate of corneal graft rejection, perhaps especially in the days immediately following surgery when risk of rejection is highest and when typical steroid eye drop administration requirements are particularly onerous.

Nanoparticles coated with high molecular weight PEG penetrate mucus and provide uniform vaginal and colorectal distribution in vivo

AIM We previously reported that nanoparticles (NPs) coated with 10 kDa PEG were mucoadhesive. Here, we demonstrate that by increasing the surface density, PEG with molecular weight (MW) as high as 40 kDa can be used as a mucoinert NP surface coating. MATERIALS & METHODS We compared two sets of reaction conditions for coating model polystyrene NPs with 10 kDa PEG and used optimized conditions to coat NPs with PEG as high as 40 kDa in MW. We then characterized NP transport in human cervicovaginal mucus ex vivo. We further administered PEG-coated NPs to the mouse cervicovaginal tract and colorectum to assess mucosal distribution in vivo. RESULTS & CONCLUSION We demonstrate here that PEG with MW as high as 40 kDa can be densely grafted to the surface of NP to prevent interactions with mucus. NP coated with 10-40 kDa PEG rapidly diffused through human cervicovaginal mucus ex vivo, and uniformly lined the mouse colorectal and vaginal epithelium in vivo.

Non-Covalent Photo-Patterning of Gelatin Matrices Using Caged Collagen Mimetic Peptides

To address the downside of conventional photo-patterning which can alter the chemical composition of protein scaffolds, we developed a non-covalent photo-patterning strategy for gelatin (denatured collagen) hydrogels that utilizes UV activated triple helical hybridization of caged collagen mimetic peptide (caged CMP). Here we present 2D and 3D photo-patterning of gelatin hydrogels enabled by the caged CMP derivatives, as well as creation of concentration gradients of CMPs. CMPs specificity for binding to gelatin allows patterning of almost any synthetic or natural gelatin-containing matrix, such as gelatin-methacrylate hydrogels and corneal tissues. This is a radically new tool for immobilizing drugs to natural tissues and for functionalizing scaffolds for complex tissue formation.

Nanoparticles that do not adhere to mucus provide uniform and long-lasting drug delivery to airways following inhalation

Debunking the mucoadhesion myth: Nonsticky particles for enhanced pulmonary drug delivery. Mucoadhesive particles (MAP) have been widely explored for pulmonary drug delivery because of their perceived benefits in improving particle residence in the lungs. However, retention of particles adhesively trapped in airway mucus may be limited by physiologic mucus clearance mechanisms. In contrast, particles that avoid mucoadhesion and have diameters smaller than mucus mesh spacings rapidly penetrate mucus layers [mucus-penetrating particles (MPP)], which we hypothesized would provide prolonged lung retention compared to MAP. We compared in vivo behaviors of variously sized, polystyrene-based MAP and MPP in the lungs following inhalation. MAP, regardless of particle size, were aggregated and poorly distributed throughout the airways, leading to rapid clearance from the lungs. Conversely, MPP as large as 300 nm exhibited uniform distribution and markedly enhanced retention compared to size-matched MAP. On the basis of these findings, we formulated biodegradable MPP (b-MPP) with an average diameter of <300 nm and examined their behavior following inhalation relative to similarly sized biodegradable MAP (b-MAP). Although b-MPP diffused rapidly through human airway mucus ex vivo, b-MAP did not. Rapid b-MPP movements in mucus ex vivo correlated to a more uniform distribution within the airways and enhanced lung retention time as compared to b-MAP. Furthermore, inhalation of b-MPP loaded with dexamethasone sodium phosphate (DP) significantly reduced inflammation in a mouse model of acute lung inflammation compared to both carrier-free DP and DP-loaded MAP. These studies provide a careful head-to-head comparison of MAP versus MPP following inhalation and challenge a long-standing dogma that favored the use of MAP for pulmonary drug delivery.

Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina

Significance Recent evidence suggests human retinal Müller glia retain a potential for neuronal regeneration. Defining the mechanisms governing retinal repair in robustly regenerative species may provide insights for harnessing this potential therapeutically. Here, we investigated roles of the innate immune system during rod photoreceptor regeneration in zebrafish. Our data establish a role for retinal microglia, the tissue-resident macrophage of the retina, in regulating retinal Müller glia responsiveness to cell death, and thereby controlling photoreceptor regeneration kinetics. Further, we show that immunosuppression can either inhibit or accelerate photoreceptor regeneration kinetics depending on the timing of treatment. We conclude that modulation of immune cell responses to retinal neuron cell death stands as a promising strategy for promoting repair of the human eye. Müller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration—i.e., selective cell-loss paradigms akin to degenerative disease—are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of (i) rod cell clearance, (ii) MG/progenitor cell proliferation, and (iii) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions.