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Purpose: The present study aimed to determine the dose-response relationship between targeted nanocarriers released from a novel, sustained release formulation and their ability to specifically deplete cells responsible for the development of posterior capsular opacification (PCO) in month-long, dynamic cell cultures. Methods: Injectable, thermosensitive poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid) triblock copolymer hydrogels were loaded with either a low or a high dose of doxorubicin-loaded antibody-targeted nanocarriers (G8:3DNA:Dox). Human rhabdomyosarcoma cells, selected for their expression of PCO marker brain-specific angiogenesis inhibitor 1 (BAI1), were kept under dynamic media flow and received either a low or high dose of nanocarriers. Cells were fixed and stained at predetermined time points to evaluate targeted depletion of BAI1+ cells. Results: A lower dose of nanocarriers in hydrogel depleted BAI1+ cells at a slower rate than the higher dose, whereas both reached over 90% BAI1+ cellular nonviability at 28 days. Both treatment groups also significantly lowered the relative abundance of BAI1+ cells in the population compared with the control group. Conclusions: Controlled release of a lower dose of nanocarriers can still achieve therapeutically relevant effects in the prevention of PCO, while avoiding potential secondary effects associated with the administration of a higher dose.
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The next generation of anticancer agents are emerging from rationally designed nanostructured materials. This work involved the synthesis and characterization of novel hollow DNA-conjugated gold nanoparticles (DNA-AuNPs) for controlled drug delivery. Polyethyleneimine (PEI) was bound to AuNPs, forming polymer-shell nanoparticles. Dissolution of the gold core via iodine formed hollow core polymeric nanoparticles (HCPNPs) and a high density (85 molecules/particle) of DNA intercalated with daunorubicin was conjugated. Particles were spherical with an average diameter of 105.7±17.3â nm and zeta potential of 20.4±3.54â mV. We hypothesize the DNA backbone electrostatically condensed to the primary amines on the surface of the particle toroidally, weaving itself within the polymer shell. During the DNA intercalation process, increasing the ionic concentration and decreasing the amine/phosphate ratio 10-fold increased drug intercalation 64 % and 61 %, respectively, allowing us to determine the optimal method of particle synthesis. As intercalation sites increased with increasing DNA strand length, drug loading increased. An average of 874±40.1 daunorubicin molecules were loaded per HCPNP. HCPNPs with drug intercalated DNA have strong potential to be clinically efficacious drug delivery vehicles due to the versatility of DNA and high drug loading capacities.
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ADN , Daunorrubicina , Oro , Polietileneimina , ADN/química , Daunorrubicina/química , Polietileneimina/química , Oro/química , Nanopartículas del Metal/química , Antineoplásicos/química , Portadores de Fármacos/química , Tamaño de la Partícula , Nanopartículas/química , HumanosRESUMEN
This study details the formation and characterisation of a novel nicotinamide adenine dinucleotide (NAD+)-associated polymeric nanoparticle system. The development of a polyelectrolyte complex (PEC) composed of two natural polyelectrolytes, hyaluronic acid and poly(L-lysine), and an evaluation of its suitability for NAD+ ocular delivery, primarily based on its physicochemical properties and in vitro release profile under physiological ocular flow rates, were of key focus. Following optimisation of formulation method conditions such as complexation pH, mode of addition, and charge ratio, the PEC was successfully formulated under mild formulation conditions via polyelectrolyte complexation. With a size of 235.1 ± 19.0 nm, a PDI value of 0.214 ± 0.140, and a zeta potential value of - 38.0 ± 1.1 mV, the chosen PEC, loaded with 430 µg of NAD+ per mg of PEC, exhibited non-Fickian, sustained release at physiological flowrates of 10.9 ± 0.2 mg of NAD+ over 14 h. PECs containing up to 200 µM of NAD+ did not induce any significant cytotoxic effects on an immortalised human corneal epithelial cell line. Using fluorescent labeling, the NAD+-associated PECs demonstrated retention within the corneal epithelium layer of a porcine model up to 6 h post incubation under physiological conditions. A study of the physicochemical behaviour of the PECs, in terms of size, zeta potential and NAD+ complexation in response to environmental stimuli,highlighted the dynamic nature of the PEC matrix and its dependence on both pH and ionic condition. Considering the successful formation of reproducible NAD+-associated PECs with suitable characteristics for ocular drug delivery via an inexpensive formulation method, they provide a promising platform for NAD+ ocular delivery with a strong potential to improve ocular health.
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Ácido Hialurónico , NAD , Humanos , Animales , Porcinos , Polielectrolitos/química , Polilisina , Sistemas de Liberación de MedicamentosRESUMEN
Research of nanoscale nucleic acid carriers has garnered attention in recent years due to their distinctive and controllable properties. However, current knowledge is limited in how we can efficiently utilize these systems for clinical applications. Several researchers have pioneered new and innovative nanocarrier drug delivery systems, but understanding physiochemical properties and behavior in vivo is vital to implementing them as clinical drug delivery platforms. In this review, we outline the most significant innovations in the synthesis, physical properties, and utilization of nucleic acid nanocarriers in the past 5 years, addressing the crucial properties which improve nanocarrier characteristics, delivery, and drug release. The challenges of controlling the transport of nucleic acid nanocarriers and therapeutic release for biological applications are outlined. Barriers which inhibit effective transport into tissue are discussed with emphasis on the modifications needed to overcome such obstacles. The novel strategies discussed in this work summarize the pivotal features of modern nucleic nanocarriers and postulate where future developments could revolutionize the translation of these tools into a clinical setting.
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Portadores de Fármacos , Ácidos Nucleicos , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Liberación de FármacosRESUMEN
Extensive research is currently being conducted into novel ocular drug delivery systems (ODDS) that are capable of surpassing the limitations associated with conventional intraocular anterior and posterior segment treatments. Nanoformulations, including those synthesised from the natural, hydrophilic glycosaminoglycan, hyaluronic acid (HA), have gained significant traction due to their enhanced intraocular permeation, longer retention times, high physiological stability, inherent biocompatibility, and biodegradability. However, conventional nanoformulation preparation methods often require large volumes of organic solvent, chemical cross-linkers, and surfactants, which can pose significant toxicity risks. We present a comprehensive, critical review of the use of HA in the field of ophthalmology and ocular drug delivery, with a discussion of the physicochemical and biological properties of HA that render it a suitable excipient for drug delivery to both the anterior and posterior segments of the eye. The pivotal focus of this review is a discussion of the formation of HA-based nanoparticles via polyelectrolyte complexation, a mild method of preparation driven primarily by electrostatic interaction between opposing polyelectrolytes. To the best of our knowledge, despite the growing number of publications centred around the development of HA-based polyelectrolyte complexes (HA-PECs) for ocular drug delivery, no review articles have been published in this area. This review aims to bridge the identified gap in the literature by (1) reviewing recent advances in the area of HA-PECs for anterior and posterior ODD, (2) describing the mechanism and thermodynamics of polyelectrolyte complexation, and (3) critically evaluating the intrinsic and extrinsic formulation parameters that must be considered when designing HA-PECs for ocular application.
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Contact lenses are one of the most successful biomaterials in history with a global market estimated to be worth over $17 billion in 2025. Silicone hydrogel contact lenses dominate the market and are complex biphasic biomaterials with several critical material properties needed for clinical use. Careful consideration of composition and chemistry is needed to identify formulations of lenses meeting all commercial standards with the potential for improved manufacturability, cost, and/or next generation use. Four silicone macromers were investigated in this work with varying symmetry of siloxane units and macromer structure, number of siloxane groups, branching, length, and concentration. Novel silicone hydrogel lenses were produced and evaluated for optical transmittance, elastic modulus, oxygen transmissibility, water content, and surface wettability. Several lenses met commercial standards and demonstrated an increase in oxygen permeability (Dk) and inverse relationship with elastic modulus and siloxane concentration, respectively. A hydrophobic/hydrophilic ratio below 1.4 was needed for a co-continuous water phase. Substitution of methoxypropyl groups for butyl groups increased hydrophobic microdomains leading to decreased optical quality and mechanical properties. Generally, fluorine-containing silicone macromers allowed for a wider range of successful compositions, and above a certain hydrophilic composition, the presence of trifluoropropyl groups resulted in improved solubility and optically clear lenses. Data also showed asymmetric siloxane macromers have potential to meet critical lens properties at lower overall siloxane content. New lens materials with wider composition ranges meeting all clinical lens properties is a significant challenge and may significantly expand the field.
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Lentes de Contacto , Siliconas , Materiales Biocompatibles/análisis , Hidrogel de Polietilenoglicol-Dimetacrilato , Hidrogeles/química , Oxígeno , Siliconas/química , Siloxanos , AguaRESUMEN
Purpose: To compare a novel, sustained release formulation and a bolus injection of a targeted nanocarrier for the ability to specifically deplete cells responsible for the development of posterior capsule opacification (PCO) in week-long, dynamic cell cultures. Methods: A novel, injectable, thermosensitive poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymer hydrogel was engineered for the sustained release of targeted, nucleic acid nanocarriers loaded with cytotoxic doxorubicin (G8:3DNA:Dox). Human rhabdomyosarcoma (RD) cells were used due to their expression of brain-specific angiogenesis inhibitor 1 (BAI1), a specific marker for the myofibroblasts responsible for PCO. Under constant media flow, nanocarriers were injected into cell cultures as either a bolus or within the hydrogel. Cells were fixed and stained every other day for 7 days to compare targeted depletion of BAI1+ cells. Results: The formulation transitions to a gel at physiological temperatures, is optically clear, noncytotoxic, and can release G8:3DNA:Dox nanocarriers for up to 4 weeks. In RD cell cultures, G8:3DNA:Dox nanocarriers specifically eliminated BAI1+ cells. The bolus nanocarrier dose showed significantly reduced cell depletion overtime, while the sustained release of nanocarriers showed increased cell depletion over time. By day 7, <2% of BAI1+ cells were depleted by the bolus injection and 74.2% BAI1+ cells were targeted by the sustained release of nanocarriers. Conclusions: The sustained release of nanocarriers from the hydrogel allows for improved therapeutic delivery in a dynamic system. This method can offer a more effective and efficient method of prophylactically treating PCO after cataract surgery.
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Opacificación Capsular , Hidrogeles , ADN , Preparaciones de Acción Retardada , Doxorrubicina , Humanos , Ácido Láctico , PolietilenglicolesRESUMEN
Nucleic acids are versatile materials capable of forming smart nanocarriers with highly controllable therapeutic delivery. DNA-gated release is a mechanism by which DNA oligonucleotides physically block the release of encapsulated drugs from porous nanoparticles. We extend this mechanism to be used with drugs bound to the surface of DNA-capped gold nanoparticles (AuNPs). We investigated DNA monolayers of different thicknesses and hybridization states to determine how DNA surface architecture can affect the release of a template drug bound to the gold surface. DNA layers are investigated on the planar gold surface via quartz crystal microbalance with dissipation and on AuNPs via dynamic light scattering. The resultant layer architectures were studied for their effect on the release rate of drugs. We observed that varying DNA architectures on AuNPs result in different release rates of the drug. The rate of drug release can be slowed using either folded or randomly coiled DNA sequences, which act as a physical barrier to diffusion. DNA monolayers with upright orientation release drugs more quickly. When the longer single-stranded DNA is used, the drug release is slowed even further. However, even upright DNA layers provide a barrier to drug diffusion at longer sequence lengths. We hypothesize that it is the architecture of the DNA layer, influenced by the folded or upright orientation of individual DNA molecules, that affects the free diffusion of the drug away from the AuNP surface. This mechanism may improve the biological availability of many surface-bound drugs on solid, DNA-capped nanoparticles.
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Nanopartículas del Metal , Ácidos Nucleicos , Oro/química , Nanopartículas del Metal/química , Hibridación de Ácido Nucleico , Tecnicas de Microbalanza del Cristal de CuarzoRESUMEN
This is the protocol for a Campbell systematic review. The objectives are as follows: to produce a mega-map which identifies, maps and provides a visual interactive display, based on systematic reviews on all the main aspects of elder abuse in both the community and in institutions, such as residential and long-term care institutions.
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Since the seminal work of Wichterle in 1965 describing the first soft contact lenses and their potential for ocular drug delivery, the field has yet to realize his vision. Maintaining all lens commercial properties combined with a mechanism for controlled drug release of therapeutically relevant concentrations for duration of wear is a major challenge. Here, successful in vivo week-long sustained release of a small molecular weight therapeutic in rabbits from extended-wear silicone hydrogel contact lenses meeting all commercial specifications by utilizing a novel macromolecular memory strategy is reported for the first time. Lens-treated eyes show a continuous, therapeutically relevant bromfenac tear concentration of 256.4 ± 23.1 µg mL-1 for 8 days. Bromday (bromfenac ophthalmic solution, 0.09%, Bausch+Lomb) topical drops exhibit a quick peak concentration of 269.3 ± 85.7 µg mL-1 and 100 min duration. Bioavailability (AUC0-8days ) and mean residence time of lenses are 26 and 155 times higher than drops, respectively. Lenses are safe, well tolerated, and no corneal histological differences are observed. This work highlights the enormous potential of drug releasing lenses as a platform strategy, and offers a new dropless clinical strategy for post-cataract, uveitis, post-LASIK, and corneal abrasion treatment.
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Lentes de Contacto de Uso Prolongado , Lentes de Contacto Hidrofílicos , Animales , Córnea , Hidrogel de Polietilenoglicol-Dimetacrilato , Hidrogeles , Conejos , SiliconasRESUMEN
Purpose: This work demonstrates seven-day controlled and extended in vitro physiological flow dual release of multiple post-ocular surgery therapeutics from extended-wear contact lenses as a dropless alternative for treatment of uveitis and corneal inflammation, pain, and infection. Lens replacement each week optimizes treatment matching patient recall time with the ability to increase or decrease dosage. Methods: Lenses were synthesized using molecular imprinting to create lenses with macromolecular memory for diclofenac sodium (DS) and dexamethasone sodium phosphate (DMSP), as well as bromfenac sodium (BS) and moxifloxacin (MOX). Drug uptake and release were analyzed, and physical properties were measured and compared to commercial standards. Results: DS + DMSP-loaded lenses demonstrated seven-days-plus release of each, whereas controls released more than 85% of their payload within the first day. Lenses loaded with BS + MOX demonstrated release of BS and MOX for 11 and eight days, respectively. Structural analysis demonstrated statistically similar mesh size and average molecular weight between crosslinks between imprinted lenses and controls, suggesting that release extension was due to formation of macromolecular memory sites rather than a tighter polymer architecture. Conclusions: Lenses demonstrated in this work have significant clinical applications as an eye drop alternative, possessing the ability to be worn continuously for one week while delivering a consistent amount of therapeutic for the duration of wear. Translational Relevance: In vitro physiological flow release results demonstrate the clinical potential of therapeutic contact lenses as a dropless vehicle for ocular drug delivery.
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Catarata , Lentes de Contacto Hidrofílicos , Oftalmología , Procedimientos Quirúrgicos Refractivos , Uveítis , Preparaciones de Acción Retardada , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Hidrogeles/uso terapéutico , Siliconas , Uveítis/tratamiento farmacológicoRESUMEN
Posterior segment eye diseases (PSEDs) including age macular degeneration (AMD) and diabetic retinopathy (DR) are amongst the major causes of irreversible blindness worldwide. Due to the numerous barriers encountered, highly invasive intravitreal (IVT) injections represent the primary route to deliver drugs to the posterior eye tissues. Thus, the potential of a more patient friendly topical route has been widely investigated. Mucoadhesive formulations can decrease precorneal clearance while prolonging precorneal residence. Thus, they are expected to enhance the chances of adherence to corneal and conjunctival surfaces and as such, enable increased delivery to the posterior eye segment. Among the mucoadhesive polymers available, chitosan is the most widely explored due to its outstanding mucoadhesive characteristics. In this review, the major PSEDs, their treatments, barriers to topical delivery, and routes of topical drug absorption to the posterior eye are presented. To enable the successful design of mucoadhesive ophthalmic drug delivery systems (DDSs), an overview of mucoadhesion, its theory, characterization, and considerations for ocular mucoadhesion is given. Furthermore, chitosan-based DDs that have been explored to promote topical drug delivery to the posterior eye segment are reviewed. Finally, challenges of successful preclinical to clinical translation of these DDSs for posterior eye drug delivery are discussed.
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Over the past half century, contact lenses have been investigated for their potential as drug delivery devices for ocular therapeutics. Hundreds of studies have been published in the pursuit of the most effective and efficient release strategies and methods for contact lens drug delivery. This paper provides a thorough overview of the various contact lens drug delivery strategies, with a specific, comprehensive focus on in vivo studies that have been published since the field began in 1965. Significant accomplishments, current trends, as well as future strategies and directions are highlighted. In vivo study analysis provides a straightforward perspective and assessment of method success and commercialization potential in comparison to benchtop, in vitro studies. Analysis of the majority of published work indicates in vitro and in vivo studies do not correlate with a correlation coefficient of 0.25, with many in vitro studies grossly overestimating drug release duration and not showing appreciable drug release control. However, there has been an increase in activity in the last decade, and some methods have generated promising results exhibiting controlled release with commercialization potential. Clinical translation of drug releasing lenses is on the horizon and has high potential to impact a large number of patients providing efficacious treatment compared to current topical treatments.
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OBJECTIVE: We asked whether letters of recommendation (LOR) written for applicants to vascular surgery (VS), a field where men have traditionally predominated, differentially highlight attributes based on applicant gender. For comparison, LOR for applicants to Obstetrics and Gynecology (Ob/Gyn), a surgical field where women are highly represented were evaluated. DESIGN: We performed a cross-sectional review of LORs for students applying to VS or Ob/Gyn at our institution from 2017 to2018. Blinded to the gender of both the applicant and the letter author, investigators assessed word count per letter and used published rubrics to quantify how many words in the following categories: communal ("friendly"), able ("competent"), standout ("exceptional"), and grindstone ("hardworking"). Frequencies were reported as a function of specialty and gender. SETTING: The study was performed at the University of Pittsburgh Medical Center and included letters written for applicants only to the stated residency programs at University of Pittsburgh Medical Center. PARTICIPANTS: LOR written for self-identified women and men applying to both residencies from US-based allopathic medical schools were de-identified and evaluated by blinded reviewers. RESULTS: One hundred and ninety-eight letters were reviewed for vascular surgery applicants. Two hundred letters were randomly selected from applications to Ob/Gyn as a comparison. Fifty-four vascular (27.8% women) and 63 Ob/Gyn (77.8% women) applicants were reviewed (p < 0.001 for gender). Licensing exam scores were higher for women than men applying to Ob/Gyn. Honor status was similar across fields and gender. Letters were shorter for VS applicants (p = 0.04). Gender-specific words (i.e., "lady" or "gentleman") were used more in VS letters (0.24 ± 0.50 vs 0.14 ± 0.42, p = 0.048). Ability words were more common (4.7 ± 2.6 vs 3.8 ± 2.1, p = 0.028) and grindstone adjectives were less common (3.4 ± 2.3 vs 4.5 ± 3.1, p = 0.024) in letters written for women compared to men VS applicants. Twenty-nine letters written for students applying to VS had honors status. While none written for women mentioned this achievement, 43% of those written for men did (p < 0.05). Letters for women applicants to Ob/Gyn contained more standout adjectives than those written for men (2.12 ± 2.2 vs 1.39 ± 1.25, p = 0.021). CONCLUSIONS: Reference letters for both specialties highlighted attributes differently depending on the gender of the applicant. Although this likely represents an unconscious process, care should be taken to limit potential biases in LOR which are "gatekeepers" to access and advancement.
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Ginecología , Internado y Residencia , Obstetricia , Estudios Transversales , Femenino , Humanos , Lingüística , Masculino , Selección de Personal , SexismoRESUMEN
The novel and unique design of self-assembled micro and nanostructures can be tailored and controlled through the deep understanding of the self-assembly behavior of amphiphilic molecules. The most commonly known amphiphilic molecules are surfactants, phospholipids, and block copolymers. These molecules present a dual attraction in aqueous solutions that lead to the formation of structures like micelles, hydrogels, and liposomes. These structures can respond to external stimuli and can be further modified making them ideal for specific, targeted medical needs and localized drug delivery treatments. Biodegradability, biocompatibility, drug protection, drug bioavailability, and improved patient compliance are among the most important benefits of these self-assembled structures for drug delivery purposes. Furthermore, there are numerous FDA-approved biomaterials with self-assembling properties that can help shorten the approval pathway of efficient platforms, allowing them to reach the therapeutic market faster. This review focuses on providing a thorough description of the current use of self-assembled micelles, hydrogels, and vesicles (polymersomes/liposomes) for the extended and controlled release of therapeutics, with relevant medical applications. FDA-approved polymers, as well as clinically and commercially available nanoplatforms, are described throughout the paper.
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We have developed a novel, nanosized drug carrier with high-therapeutic payload, controllable release, and the potential for active tumor targeting. It consists of a 15 nm gold nanoparticle with dense surface loading of DNA duplexes. We utilize the natural intercalating behavior of daunomycin to load the drug between DNA base pairs. We obtained a high-therapeutic payload of >1,000 drug molecules per gold nanoparticle (AuNP), one of the highest loadings reported in literature to date. We have engineered unique DNA sequences to control release of daunomycin for over 48 hr and show higher cell death compared to equivalent concentrations of free daunomycin. We have also explored cell internalization mechanisms to identify the pathways by which our gold nanoparticles enter the cell. This nanocarrier is in the ideal size range of 16-100 nm in diameter to utilize the enhanced permeability and retention effect for passive targeting to tumors. Our AuNP platform is effective as a therapeutic drug delivery device and can easily incorporate any aptamer of choice through complementary base pairing. Our work has produced an innovative nanoscale drug-delivery platform potentially leading to personalized cancer therapies through careful selection of aptamers and an adjustable drug release profile.
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Antibióticos Antineoplásicos/administración & dosificación , ADN/química , Daunorrubicina/administración & dosificación , Preparaciones de Acción Retardada/química , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacología , Aptámeros de Nucleótidos/química , Supervivencia Celular/efectos de los fármacos , Daunorrubicina/farmacocinética , Daunorrubicina/farmacología , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Humanos , Células MCF-7 , Neoplasias/tratamiento farmacológicoRESUMEN
Purpose: Cataracts are the leading cause of blindness worldwide, resulting in over 30 million surgeries each year. These cases are expected to double within the next 10 years. About 25% of all patients develop secondary cataracts or posterior capsule opacification (PCO) postsurgery. PCO is a vision impairment disorder that develops from myofibroblasts migration and contraction that deforms the capsule surrounding the lens. Currently, Nd:YAG laser therapy is used to treat PCO; however, laser is not available worldwide and adverse side effects may arise. Thus, there is a considerable unmet need for more efficacious and convenient preventive treatments for PCO. Our work focuses on engineering an innovative, prophylactic sustained release platform for DNA-based nanocarriers to further reduce the incidence of PCO. Methods: Novel, optically clear, self-assembled poly(d,l-lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-PEG) triblock copolymer hydrogels were used for the sustained release of the DNA-based nanocarriers (3DNA®) loaded with cytotoxic doxorubicin (DOX) and targeted with a monoclonal antibody called G8 (3DNA:DOX:G8), which is specific to cells responsible for PCO. Results: The 29 (w/v)% polymer hydrogels with the 3DNA nanocarriers presented over 80% of light transmittance, soft mechanical properties (<350 Pa), and sustained release for 1 month. Conclusions: In this work, we show for the first time that the hydrophobic PLGA-PEG-PLGA hydrogels can be used as platforms for sustained delivery of nucleic acid-based nanocarriers. This work demonstrates that polymeric formulations can be used for the extended delivery of ocular therapeutics and other macromolecules to treat a variety of ocular conditions.
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Antibióticos Antineoplásicos/farmacocinética , Opacificación Capsular/prevención & control , Doxorrubicina/farmacocinética , Portadores de Fármacos/química , Hidrogeles/química , Nanotecnología/métodos , Polietilenglicoles/química , Poliglactina 910/química , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/uso terapéutico , Opacificación Capsular/epidemiología , Doxorrubicina/administración & dosificación , Doxorrubicina/uso terapéutico , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Humanos , Incidencia , Micelas , Polietilenglicoles/metabolismo , Poliglactina 910/metabolismoRESUMEN
The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a remarkable circa-24 h oscillation of KaiC phosphorylation that persists for many days in vitro. Here we use high-speed atomic force microscopy (HS-AFM) to visualize in real time and quantify the dynamic interactions of KaiA with KaiC on sub-second timescales. KaiA transiently interacts with KaiC, thereby stimulating KaiC autokinase activity. As KaiC becomes progressively more phosphorylated, KaiA's affinity for KaiC weakens, revealing a feedback of KaiC phosphostatus back onto the KaiA-binding events. These non-equilibrium interactions integrate high-frequency binding and unbinding events, thereby refining the period of the longer term oscillations. Moreover, this differential affinity phenomenon broadens the range of Kai protein stoichiometries that allow rhythmicity, explaining how the oscillation is resilient in an in vivo milieu that includes noise. Therefore, robustness of rhythmicity on a 24-h scale is explainable by molecular events occurring on a scale of sub-seconds.
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Proteínas Bacterianas/metabolismo , Proteínas CLOCK/metabolismo , Ritmo Circadiano/fisiología , Simulación por Computador , Cinética , Microscopía de Fuerza Atómica , Proteínas Mutantes/metabolismo , Fosforilación , Unión Proteica , Multimerización de Proteína , Synechococcus/metabolismoRESUMEN
AIM: Polymersomes are created to deliver an enzyme-based therapy to the brain in lysosomal storage disease patients. MATERIALS & METHODS: Polymersomes are formed via the injection method using poly(ethylene glycol)-b-poly(lactic acid) (PEGPLA) and bound to apolipoprotein E, to create a brain-targeted delivery vehicle. RESULTS: Polymersomes have a smallest average diameter of 145 ± 21 nm and encapsulate ß-galactosidase at 72.0 ± 12.2% efficiency. PEGPLA polymersomes demonstrate limited release at physiologic pH (7.4), with a burst release at the acidic pH (4.8) of the lysosome. PEGPLA polymersomes facilitate delivery of active ß-galactosidase to an in vitro model of GM1 gangliosidosis. CONCLUSION: The foundation has been laid for testing of PEGPLA polymersomes to deliver enzymatic treatments to the brain in lysosomal storage disorders for the first time.
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Portadores de Fármacos/química , Terapia de Reemplazo Enzimático/métodos , Lactatos/química , Polietilenglicoles/química , beta-Galactosidasa/farmacología , Encéfalo/metabolismo , Línea Celular , Liberación de Fármacos , Gangliosidosis GM1/tratamiento farmacológico , Humanos , Concentración de Iones de Hidrógeno , Nanopartículas/química , Tamaño de la Partícula , Permeabilidad , Propiedades de SuperficieRESUMEN
Approximately 1 in 5000-8000 children are born annually with a lysosomal storage disease (LSD), which affects their cells' ability to break down naturally occurring substrates. Accumulation, or "storage," of undegraded substrates leads to a wide variety of clinical symptoms, and early mortality. Currently, for LSDs with central nervous system (CNS) involvement, there is no available treatment. Four methods of treatment are being explored in clinical trials and preclinical settings: enzyme replacement therapy, ex vivo gene therapy, in vivo gene therapy, and nanoparticle-based therapy. In general, each therapeutic approach has been hindered by an inability to cross the blood-brain barrier (BBB) without invasive intracranial surgeries. Also, once the treatment has entered the brain, it is difficult to ensure therapeutic levels of enzyme distributed evenly throughout the entire parenchyma. Enzyme replacement therapy (ERT) is the current standard of care for lysosomal diseases without CNS involvement. However, with the recent advent of nanoparticle-based therapy, direct targeting of either gene therapy or ERT to the brain has become plausible. Ex vivo gene therapy, in vivo gene therapy, ERT and nanoparticle-based therapies are explained, while synthesizing and analyzing their potential as clinical treatments targeted to the CNS. While difficulties in treating the entire brain remain, preclinical studies demonstrate profound therapeutic benefit in animal models and generate hope for successful translation to humans.