Pretreatment of human cervicovaginal mucus with pluronic F127 enhances nanoparticle penetration without compromising mucus barrier properties to herpes simplex virus.

Mucosal drug delivery nanotechnologies are limited by the mucus barrier that protects nearly all epithelial surfaces not covered with skin. Most polymeric nanoparticles, including polystyrene nanoparticles (PS), strongly adhere to mucus, thereby limiting penetration and facilitating rapid clearance from the body. Here, we demonstrate that PS rapidly penetrate human cervicovaginal mucus (CVM), if the CVM has been pretreated with sufficient concentrations of Pluronic F127. Importantly, the diffusion rate of large polyethylene glycol (PEG)-coated, nonmucoadhesive nanoparticles (PS-PEG) did not change in F127-pretreated CVM, implying that F127 did not significantly alter the native pore structure of CVM. Additionally, herpes simplex virus type 1 (HSV-1) remains adherent in F127-pretreated CVM, indicating that the presence of F127 did not reduce adhesive interactions between CVM and the virions. In contrast to treatment with a surfactant that has been approved for vaginal use as a spermicide (nonoxynol-9 or N9), there was no increase in inflammatory cytokine release in the vaginal tract of mice after daily application of 1% F127 for 1 week. Pluronic F127 pretreatment holds potential as a method to safely improve the distribution, retention, and efficacy of nanoparticle formulations without compromising CVM barrier properties to pathogens.

Cone-beam CT evaluation of post-extraction alveolar bone changes at the maxillary incisor sites in an East Asian population: A cross-sectional study.

Objective: Understanding the characteristics of alveolar bone resorption in an East Asian population after maxillary incisor extraction and providing a reference for implant treatment plans. Study design: Cone-beam computerized tomography (CBCT) data of 125 East Asian patients with unilateral extraction of maxillary incisors for 3 months were collected. The alveolar bone width and height in the extraction sites were measured and compared with the corresponding contralateral sites. Results: The differences in alveolar bone width between the extraction site and contralateral site were as follows: 4.11 mm, 2.68 mm, and 2.09 mm (3 mm, 5 mm, 7 mm apical from CEJ of the contralateral tooth). Data are expressed as the median. The horizontal resorption ratio of alveolar bone was 49.94 %, 31.5 %, and 24.46 %. The difference in alveolar bone height was 0.78 mm. The vertical resorption ratio was 7.78 %. The resorption did not differ significantly between sexes and was not significantly affected by tooth positions. Conclusions: In the studied East Asian population, significant horizontal and vertical alveolar bone resorption occurs after natural healing of maxillary incisor extraction for 3 months. The closer to the alveolar ridge crest, the more significant the horizontal resorption, resulting in an "inverted triangle" shape residual alveolar bone.

Efficacy of Electronic Cigarettes vs Varenicline and Nicotine Chewing Gum as an Aid to Stop Smoking: A Randomized Clinical Trial.

Importance: Electronic cigarettes (ECs) are often used by smokers as an aid to stopping smoking, but evidence is limited regarding their efficacy compared with nicotine replacement therapy (NRT), and no evidence is available on how their efficacy compares with that of varenicline. Objective: To evaluate whether ECs are superior to NRT and noninferior to varenicline in helping smokers quit. Design, Setting, and Participants: This was a randomized clinical trial conducted at 7 sites in China and including participants who were smoking at least 10 cigarettes per day and motivated to quit, not using stop-smoking medications or EC, and willing to use any of the study products. Participants were first recruited in May 2021, and data analysis was conducted in December 2022. Interventions: A cartridge-based EC (30 mg/mL nicotine salt for 2 weeks and 50 mg/mL after that), varenicline (0.5 mg, once a day for 3 days; 0.5 mg, twice a day for 4 days; and 1 mg, twice a day, after that), and 2 mg (for smokers of ≤20 cigarettes per day) or 4 mg (>20 cigarettes per day) nicotine chewing gum, all provided for 12 weeks and accompanied by minimal behavioral support (an invitation to join a self-help internet forum). Main Outcomes and Measures: The primary outcome was sustained abstinence from smoking at 6 months as validated by an expired-air carbon monoxide reading (<8 parts per million). Participants lost to follow-up were included as nonabstainers. Results: Of 1068 participants, 357 (33.5%) were female, and the mean (SD) age was 33.9 (3.1) years. A total of 409 (38.3%), 409 (38.3%), and 250 (23.4%) participants were randomized to the EC, varenicline, and NRT arms, respectively. The 6-month biochemically validated abstinence rates were 15.7% (n = 64), 14.2% (n = 58), and 8.8% (n = 22) in the EC, varenicline, and NRT study arms, respectively. The quit rate in the EC arm was noninferior to the varenicline arm (absolute risk reduction, 1.47%; 95% CI, -1.41% to 4.34%) and higher than in the NRT arm (odds ratio, 1.92; 95% CI, 1.15-3.21). Treatment adherence was similar in all study arms during the initial 3 months, but 257 participants (62.8%) in the EC arm were still using ECs at 6 months, with no further use in the 2 other study arms. The most common adverse reactions were throat irritation (32 [7.8%]) and mouth irritation (28 [6.9%]) in the EC arm, nausea (36 [8.8%]) in the varenicline arm, and throat irritation (20 [8.0%]) and mouth irritation (22 [8.8%]) in the NRT arm. No serious adverse events were recorded. Conclusions and Relevance: The results of this randomized clinical trial found that when all treatments were provided with minimal behavior support, the efficacy of EC was noninferior to varenicline and superior to nicotine chewing gum. Trial Registration: Chinese Clinical Trial Registry: ChiCTR2100048156.

The microstructure and bulk rheology of human cervicovaginal mucus are remarkably resistant to changes in pH.

The protective barrier, lubricant, and clearance functions of mucus are intimately coupled to its microstructure and bulk rheology. Mucus gels consist of a network of mucin biopolymers along with lipids, salts, and other proteins and exhibit similar biochemical and physical properties across diverse mucosal surfaces. Nevertheless, mucus is exposed to a broad range of pH values throughout the human body. Protein functions are typically sensitive to small changes in pH, and prior investigations using reconstituted, purified mucin gels suggested mucus undergoes a transition from a low-viscosity liquid at neutral pH to a highly viscoelastic solid at low pH. We sought to determine whether those observations hold for fresh, minimally perturbed human mucus ex vivo by using different-sized muco-inert nanoparticles to probe microstructure and cone-and-plate rheometry to measure bulk rheology. We demonstrate that both the microstructure and bulk rheology of fresh, undiluted, and minimally perturbed cervicovaginal mucus exhibit relatively minor changes from pH 1-2 to 8-9, in marked contrast with the pH sensitivity of purified mucin gels. Our work also suggests additional components in mucus secretions, typically eliminated during mucin purification and reconstitution, may play an important role in maintaining the protective properties of mucus.

Nanoparticles reveal that human cervicovaginal mucus is riddled with pores larger than viruses.

The mechanisms by which mucus helps prevent viruses from infecting mucosal surfaces are not well understood. We engineered non-mucoadhesive nanoparticles of various sizes and used them as probes to determine the spacing between mucin fibers (pore sizes) in fresh undiluted human cervicovaginal mucus (CVM) obtained from volunteers with healthy vaginal microflora. We found that most pores in CVM have diameters significantly larger than human viruses (average pore size 340 +/- 70 nm; range approximately 50-1800 nm). This mesh structure is substantially more open than the 15-100-nm spacing expected assuming mucus consists primarily of a random array of individual mucin fibers. Addition of a nonionic detergent to CVM caused the average pore size to decrease to 130 +/- 50 nm. This suggests hydrophobic interactions between lipid-coated "naked" protein regions on mucins normally cause mucin fibers to self-condense and/or bundle with other fibers, creating mucin "cables" at least three times thicker than individual mucin fibers. Although the native mesh structure is not tight enough to trap most viruses, we found that herpes simplex virus (approximately 180 nm) was strongly trapped in CVM, moving at least 8,000-fold slower than non-mucoadhesive 200-nm nanoparticles. This work provides an accurate measurement of the pore structure of fresh, hydrated ex vivo CVM and demonstrates that mucoadhesion, rather than steric obstruction, may be a critical protective mechanism against a major sexually transmitted virus and perhaps other viruses.

Biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier.

Protective mucus coatings typically trap and rapidly remove foreign particles from the eyes, gastrointestinal tract, airways, nasopharynx, and female reproductive tract, thereby strongly limiting opportunities for controlled drug delivery at mucosal surfaces. No synthetic drug delivery system composed of biodegradable polymers has been shown to penetrate highly viscoelastic human mucus, such as non-ovulatory cervicovaginal mucus, at a significant rate. We prepared nanoparticles composed of a biodegradable diblock copolymer of poly(sebacic acid) and poly(ethylene glycol) (PSA-PEG), both of which are routinely used in humans. In fresh undiluted human cervicovaginal mucus (CVM), which has a bulk viscosity approximately 1,800-fold higher than water at low shear, PSA-PEG nanoparticles diffused at an average speed only 12-fold lower than the same particles in pure water. In contrast, similarly sized biodegradable nanoparticles composed of PSA or poly(lactic-co-glycolic acid) (PLGA) diffused at least 3,300-fold slower in CVM than in water. PSA-PEG particles also rapidly penetrated sputum expectorated from the lungs of patients with cystic fibrosis, a disease characterized by hyperviscoelastic mucus secretions. Rapid nanoparticle transport in mucus is made possible by the efficient partitioning of PEG to the particle surface during formulation. Biodegradable polymeric nanoparticles capable of overcoming human mucus barriers and providing sustained drug release open significant opportunities for improved drug and gene delivery at mucosal surfaces.

Expressions of cytokeratin 14 and proliferating cell nuclear antigen in the Hertwig's epithelial root sheath of a Vps4b knockout mouse.

OBJECTIVES: The effect of Vps4b gene mutation on the expressions of cytokeratin 14 (CK14) and proliferating cell nuclear antigen (PCNA) in the Hertwig's epithelial root sheath (HERS) is investigated. METHODS: The bilateral mandibular tissues of mouse on postnatal days 5, 9, 11, 15, and 19 were removed. The mandibular first molar tissue sections were obtained after paraffin embedding. The CK14 and PCNA expressions in the epithelial root sheath of the normal mouse and Vps4b knockout mouse were compared through immunohistochemistry. RESULTS: On postnatal day 5, the normal mouse began to form HERS and had a strong positive PCNA expression in the HERS cells; on postnatal day 9, the HERS structure was continuous, and PCNA was positive in the HERS cells; on postnatal day 11, a small portion of HERS began to break, and PCNA was weakly positive in the HERS cells; on postnatal day 15, HERS continued to fracture; PCNA was weakly and positively expressed in the HERS cells on the root surface; on postnatal day 19, the tooth root reached normal physiological length, and PCNA was positively expressed in the HERS cells of the terminal part. Similar to the normal mouse, the gene knockout mouse also formed a HERS structure on postnatal day 5. However, HERS began to break on postnatal day 9. On postnatal day 19, only a few fragments of HERS were found on the root surface, and the root development was immature. Moreover, the expression intensity of PCNA in the gene knockout mouse was decreased. CONCLUSIONS: The Vps4b gene mutation may change the CK14 and PCNA expressions, leading to abnormal root development.

Development of a Uricase-Free Colorimetric Biosensor for Uric Acid Based on PPy-Coated Polyoxometalate-Encapsulated Fourfold Helical Metal-Organic Frameworks.

Developing a new cost-effective and reliable approach used for the detection of uric acid (UA) with no requirement of uricase is still very challenging. Herein, an easily realized, cost-effective, and uricase-free approach is reported for selective colorimetric biosensing of UA utilizing polypyrrole (PPy)-coated polyoxometalate-encapsulated fourfold helical metal-organic frameworks Ag5[bimt]2[PMo12O40]·2H2O (Ag5PMo12) as a monolithic peroxidase mimic. It is demonstrated that the as-obtained Ag5PMo12@PPy possesses excellent peroxidase-like activity originated from the synergistic effect to induce catalytic oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to green oxTMB in the presence of H2O2. Then, the green oxTMB can be selectively converted to colorless TMB induced by UA; thus, UA can inhibit the catalytic oxidation of TMB. Based on these results, a uricase-free colorimetric biosensor for UA is achieved with a linear detection range of 1-50 µM and a detection limit of 0.47 µM. More importantly, the developed biosensor is suited for simple-operated and good reliable UA detection in clinical samples, showing promising application ability in clinical diagnosis and relative fields.

[Spatio-temporal expression of dentin sialophosphoprotein and collagen â during molar tooth germ development in vps4b knockout mouse].

OBJECTIVE: To verify the effect of the mutant gene vps4b on the expression of tooth development-related proteins, dentin sialophosphoprotein (DSPP) and collagenⅠ (COL-Ⅰ). METHODS: Paraffin tissue sections of the first molar tooth germ were obtained from the heads of fetal mice at the embryonic stages of 13.5, 14.5, and 16.5 days and from the mandibles of larvae aged 2.5 and 7 days after birth. The immunohistochemical method was used to detect the expression and location of DSPP and COL-Ⅰ in wild-type mouse and vps4b knockout mouse. RESULTS: DSPP and COL-Ⅰ were not found in the bud and cap stages of wild-type mouse molar germ. In the bell stage, DSPP was positively expressed in the inner enamel epithelium and dental papilla, whereas COL-Ⅰ was strongly expressed in the dental papilla and dental follicle. During the secretory and mineralized periods, DSPP and COL-Ⅰ were intensely observed in ameloblasts, odontoblasts, and dental follicles, but COL-Ⅰ was also expressed in the dental papilla. After vps4b gene knockout, DSPP was not expressed in the dental papilla of the bell stage and in the dental papilla and dental follicle of the secretory phase. The expression position of COL-Ⅰ in the bell and mineralization phase was consistent with that in the wild-type mice. Moreover, the expression of COL-Ⅰ in the dental papilla changed in the secretory stage. CONCLUSIONS: Gene vps4b plays a significant role in the development of tooth germ. The expression of DSPP and COL-Ⅰ may be controlled by gene vps4b and regulates the development of tooth dentin and cementum together with vps4b.

Antifertility effectiveness of a novel polymer matrix composite and its influence on the endometrium in rhesus macaques (Macaca mulatta).

OBJECTIVE(S): To explored the antifertility effectiveness and influence on the endometrium of a micro-copper/low-density polyethylene/methyl vinyl silicone rubber (Cu/LDPE/MVQ) composite in rhesus macaques. STUDY DESIGN: Healthy reproductive aged female rhesus macaques underwent abdominal hysterotomy for surgical placement of either the experimental Cu/LDPE/MVQ composite (Cu/LDPE/MVQ, n=5), bare copper wire (Cu, n=5), or hysterotomy only sham-operation group [(SOI, n=4), (SOII, n=6)]. Females in the Cu/LPDE/MVQ, Cu, and SOI groups were housed with fertile males for approximately three menstrual cycles. We assessed pregnancy by hysterectomy. Females in the Cu/LDPE/MVQ, Cu, and SOII groups underwent hysterectomy at about 4 months post-insertion for histologic assessment of morphologic changes of the endometrium, evaluation of materials using scanning electron microscopy (SEM), and evaluation of the inflammatory markers, including substance P receptor (SPR), associated with endometrial bleeding using enzyme linked immunosorbent assay, quantitative RT-PCR, and Western blot analyses. RESULTS: All of the SOI group females became pregnant (4/4, 100%). In contrast, no pregnancies occurred in either the Cu/LDPE/MVQ (0/5, 0%) or Cu (0/5, 0%) groups. We observed histologic features consistent with chronic endometrial inflammation in all females of the Cu group, but none of the SOII or Cu/LDPE/MVQ animals. Levels of inflammatory markers were significantly increased in the Cu group, compared with SOII or Cu/LDPE/MVQ groups (p<.05). SEM showed evidence of corrosion in the Cu wire not seen in the Cu/LDPE/MVQ group. CONCLUSION(S): Cu/LDPE/MVQ material provided a contraceptive effect similar to Cu in macaques, with a lower impact on inflammation and inflammatory markers of the endometrium. IMPLICATIONS: This study demonstrates the possibility of a Cu/LDPE/MVQ composite as an alternative to conventional copper device materials.

[Effect of On-line NaClO Backwashing on Microbial Communities in an Inverted A2O-MBR System].

To study the effect of on-line NaClO backwashing on the microbial community structure of membrane bioreactor (MBR) systems, a reversed A2O-MBR reactor was used to monitor system performance, membrane fouling, and microbial community structure. Measurements were made during the stabilization stage, the on-line pure water backwashing stage, and the on-line NaClO backwashing stage. The results showed that key system performance indicators during NaClO backwashing stage, such as COD, ammonia nitrogen, and TN treatment efficiency, were similar to previous stages. The average membrane fouling rate during the on-line pure water backwashing period was lower than that of the stable stage, while the membrane fouling rate increased during the on-line NaClO backwashing stage, and EPS concentration was the highest and membrane fouling was aggravated. The results of the Chao index, Simpson index, and Shannon index showed that the microbial diversity in the aerobic tank sludge remained almost stable after on-line NaClO backwashing, while the abundance of cake layer sludge increased slightly, but the microbial diversity decreased significantly. Proteobacteria was the main microbial phylum in both the aerobic tank and cake layer sludge, followed by Bacteroidetes. After on-line NaClO backwashing, the relative abundance of Proteobacteria and Bacteroides in the aerobic tank sludge did not change notably, but the composition of filter cake sludge changed significantly. Proteobacteria that possess resistance to chlorine disinfectant increased from 53.4% to 77.8%, while Bacteroides decreased from 33.44% to 14.5%. After on-line NaClO backwashing, the composition of the microbial community in aerobic tank and cake layer was similar. Azospira and Comamonadaceaea also increased significantly after NaClO backwashing. Microbial species that can tolerate NaClO treatment may be the main cause of membrane fouling.

CS/PAA@TPGS/PLGA nanoparticles with intracellular pH-sensitive sequential release for delivering drug to the nucleus of MDR cells.

Development of novel nano-drug delivery systems (NDDS) that can transport anticancer drugs into cell nuclei is still a highly desirable strategy for reversing multi-drug resistance (MDR) in cancer therapy. Herein, we designed and prepared a novel NDDS, designated S@L NPs, in which several smaller nanoparticles are contained within a larger nanoparticle. Our S@L NPs (CS/PAA/VP-16@TPGS/PLGA NPs) possess a structure in which smaller nanoparticles (Chitosan-Poly(acrylic acid) nanoparticles, CS/PAA NPs) containing the drug etoposide (VP-16) are loaded within a larger nanoparticle (Vitamin E d-a-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles, TPGS/PLGA NPs). The system utilizes intracellular pH gradients to achieve pH-sensitive sequential release within different intracellular domains of MDR cells. S@L NPs could be triggered to degrade and release CS/PAA/VP-16 NPs in the acid environment of the cytosol, endosomes or lysosomes, and CS/PAA/VP-16 NPs were capable of entering the nucleus through nucleopores. It is significant that CS/PAA/VP-16 NPs exhibit disaggregation in the alkaline environment of the nucleus and thereby release the contained anticancer drug. Further mechanistic studies showed that CS/PAA/VP-16 NPs escaped retention and degradation within lysosomes and protected the drug from P-glycoprotein-induced efflux. Simultaneously, S@L NPs enhanced the anticancer effect of the loaded drug by inducing autophagy and apoptosis of MDR cells. This novel NDDS may provide a promising platform for nuclear drug delivery for reversing MDR.

Anti-PEG antibodies alter the mobility and biodistribution of densely PEGylated nanoparticles in mucus.

UNLABELLED: Antibodies that specifically bind polyethylene glycol (PEG) can lead to rapid elimination of PEGylated therapeutics from the systemic circulation. We have recently shown that virus-binding IgG can immobilize viruses in mucus via multiple low-affinity crosslinks between IgG and mucins. However, it remains unclear whether anti-PEG antibodies in mucus may also alter the penetration and consequently biodistribution of PEGylated nanoparticles delivered to mucosal surfaces. We found that both anti-PEG IgG and IgM can readily bind nanoparticles that were densely coated with PEG polymer to minimize adhesive interactions with mucus constituents. Addition of anti-PEG IgG and IgM into mouse cervicovaginal mucus resulted in extensive trapping of mucus-penetrating PEGylated nanoparticles, with the fraction of mobile particles reduced from over 95% to only 34% and 7% with anti-PEG IgG and IgM, respectively. Surprisingly, we did not observe significant agglutination induced by either antibody, suggesting that particle immobilization is caused by adhesive crosslinks between mucin fibers and IgG or IgM bound to individual nanoparticles. Importantly, addition of corresponding control antibodies did not slow the PEGylated nanoparticles, confirming anti-PEG antibodies specifically bound to and trapped the PEGylated nanoparticles. Finally, we showed that trapped PEGylated nanoparticles remained largely in the luminal mucus layer of the mouse vagina even when delivered in hypotonic formulations that caused untrapped particles to be drawn by the flow of water (advection) through mucus all the way to the epithelial surface. These results underscore the potential importance of elucidating mucosal anti-PEG immune responses for PEGylated therapeutics and biomaterials applied to mucosal surfaces. STATEMENT OF SIGNIFICANCE: PEG, generally considered a 'stealth' polymer, is broadly used to improve the circulation times and therapeutic efficacy of nanomedicines. Nevertheless, there is increasing scientific evidence that demonstrates both animals and humans can generate PEG-specific antibodies. Here, we show that anti-PEG IgG and IgM can specifically immobilize otherwise freely diffusing PEG-coated nanoparticles in fresh vaginal mucus gel ex vivo by crosslinking nanoparticles to the mucin mesh, and consequently prevent PEG-coated nanoparticles from accessing the vaginal epithelium in vivo. Given the increasing use of PEG coatings to enhance nanoparticle penetration of mucosal barriers, our findings demonstrate that anti-PEG immunity may be a potential concern not only for systemic drug delivery but also for mucosal drug delivery.

Food emulsifier polysorbate 80 increases intestinal absorption of di-(2-ethylhexyl) phthalate in rats.

The aim of the present research was to explore whether food emulsifier polysorbate 80 can enhance the absorption of di-(2-ethylhexyl) phthalate (DEHP) and its possible mechanism. We established the high-performance liquid chromatography (HPLC) method for detecting DEHP and its major metabolite, mono-ethylhexyl phthalate (MEHP) in rat plasma, and then examined the toxicokinetic and bioavailability of DEHP with or without polysorbate 80 in rats. The study of its mechanism to increase the absorption of phthalates demonstrated that polysorbate 80 can induce mitochondrial dysfunction in time- and concentration-dependence manners in Caco-2 cells by reducing mitochondrial membrane potential, diminishing the production of the adenosine triphosphate, and decreasing the activity of electron transport chain. Our results indicated that food emulsifier applied in relatively high concentrations in even the most frequently consumed foods can increase the absorption of DEHP, and its role may be related to the structure and function damages of mitochondria in enterocytes.

Nanoparticle penetration of human cervicovaginal mucus: the effect of polyvinyl alcohol.

Therapeutic nanoparticles must rapidly penetrate the mucus secretions lining the surfaces of the respiratory, gastrointestinal and cervicovaginal tracts to efficiently reach the underlying tissues. Whereas most polymeric nanoparticles are highly mucoadhesive, we previously discovered that a dense layer of low MW polyethylene glycol (PEG) conferred a sufficiently hydrophilic and uncharged surface to effectively minimize mucin-nanoparticle adhesive interactions, allowing well-coated particles to rapidly diffuse through human mucus. Here, we sought to investigate the influence of surface coating by polyvinyl alcohol (PVA), a relatively hydrophilic and uncharged polymer routinely used as a surfactant to formulate drug carriers, on the transport of nanoparticles in fresh human cervicovaginal mucus. We found that PVA-coated polystyrene (PS) particles were immobilized, with speeds at least 4000-fold lower in mucus than in water, regardless of the PVA molecular weight or incubation concentration tested. Nanoparticles composed of poly(lactide-co-glycolide) (PLGA) or diblock copolymers of PEG-PLGA were similarly immobilized when coated with PVA (slowed 29,000- and 2500-fold, respectively). PVA coatings could not be adequately removed upon washing, and the residual PVA prevented sufficient coating with Pluronic F127 capable of reducing particle mucoadhesion. In contrast to PVA-coated particles, the similar sized PEG-coated formulations were slowed only ~6- to 10-fold in mucus compared to in water. Our results suggest that incorporating PVA in the particle formulation process may lead to the formation of mucoadhesive particles for many nanoparticulate systems. Thus, alternative methods for particle formulation, based on novel surfactants or changes in the formulation process, should be identified and developed in order to produce mucus-penetrating particles for mucosal applications.

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.

A poly(ethylene glycol)-based surfactant for formulation of drug-loaded mucus penetrating particles.

Mucosal surfaces are protected by a highly viscoelastic and adhesive mucus layer that traps most foreign particles, including conventional drug and gene carriers. Trapped particles are eliminated on the order of seconds to hours by mucus clearance mechanisms, precluding sustained and targeted drug and nucleic acid delivery to mucosal tissues. We have previously shown that polymeric coatings that minimize adhesive interactions with mucus constituents lead to particles that rapidly penetrate human mucus secretions. Nevertheless, a particular challenge in formulating drug-loaded mucus penetrating particles (MPP) is that many commonly used surfactants are either mucoadhesive, or do not facilitate efficient drug encapsulation. We tested a novel surfactant molecule for particle formulation composed of Vitamin E conjugated to 5 kDa poly(ethylene glycol) (VP5k). We show that VP5k-coated poly(lactide-co-glycolide) (PLGA) nanoparticles rapidly penetrate human cervicovaginal mucus, whereas PLGA nanoparticles coated with polyvinyl alcohol or Vitamin E conjugated to 1 kDa PEG were trapped. Importantly, VP5k facilitated high loading of paclitaxel, a frontline chemo drug, into PLGA MPP, with controlled release for at least 4 days and negligible burst release. Our results offer a promising new method for engineering biodegradable, drug-loaded MPP for sustained and targeted delivery of therapeutics at mucosal surfaces.