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Enzyme-based therapy has garnered significant attention for its current applications in various diseases. Despite the notable advantages associated with the use of enzymes as therapeutic agents, that could have high selectivity, affinity, and specificity for the target, their application faces challenges linked to physico-chemical and pharmacological properties. These limitations can be addressed through the encapsulation of enzymes in nanoplatforms as a comprehensive solution to mitigate their degradation, loss of activity, off-target accumulation, and immunogenicity, thus enhancing bioavailability, therapeutic efficacy, and circulation time, thereby reducing the number of administrations, and ameliorating patient compliance. The exploration of novel nanomedicine-based enzyme therapeutics for the treatment of challenging diseases stands as a paramount goal in the contemporary scientific landscape, but even then it is often not enough. Combining an enzyme with another therapeutic (e.g., a small molecule, another enzyme or protein, a monoclonal antibody, or a nucleic acid) within a single nanocarrier provides innovative multidrug-integrated therapy and ensures that both the actives arrive at the target site and exert their therapeutic effect, leading to synergistic action and superior therapeutic efficacy. Moreover, this strategic approach could be extended to gene therapy, a field that nowadays has gained increasing attention, as enzymes acting at genomic level and nucleic acids may be combined for synergistic therapy. This multicomponent therapeutic approach opens opportunities for promising future developments. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.
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Terapia Enzimática , Nanomedicina , Humanos , AnimalesRESUMEN
Since its discovery as one of the main components of cannabis and its affinity towards the cannabinoid receptor CB1, serving as a means to exert its psychoactivity, Δ9-tetrahydrocannabinol (Δ9-THC) has inspired medicinal chemists throughout history to create more potent derivatives. Initially, the goal was to synthesize chemical probes for investigating the molecular mechanisms behind the pharmacology of Δ9-THC and finding potential medical applications. The unintended consequence of this noble intent has been the proliferation of these compounds for recreational use. This review comprehensively covers the most exhaustive number of THC-like cannabinoids circulating on the recreational market. It provides information on the chemistry, synthesis, pharmacology, analytical assessment, and experiences related to the psychoactive effects reported by recreational users on online forums. Some of these compounds can be found in natural cannabis, albeit in trace amounts, while others are entirely artificial. Moreover, to circumvent legal issues, many manufacturers resort to semi-synthetic processes starting from legal products extracted from hemp, such as cannabidiol (CBD). Despite the aim to encompass all known THC-like molecules, new species emerge on the drug users' pipeline each month. Beyond posing a significantly high public health risk due to unpredictable and unknown side effects, scientific research consistently lags behind the rapidly evolving recreational market.
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Purpose: In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity. Methods: The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical "post-formulation" DNA addition vs "pre" addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake. Results: Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 µg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 µg/mL of pDNA with the "post" method, while maintaining their characteristics up to 100 µg/mL of pDNA with the "pre" addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 µg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes. Conclusion: In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy.
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Cationes , ADN , Plásmidos , Plásmidos/administración & dosificación , Plásmidos/química , Humanos , Cationes/química , ADN/química , ADN/administración & dosificación , Terapia Genética/métodos , Microfluídica/métodos , Tamaño de la Partícula , Nanomedicina , COVID-19/prevención & control , Liposomas/química , Transfección/métodos , Nanopartículas/química , SARS-CoV-2 , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/química , Compuestos de Amonio Cuaternario/química , Ácidos Grasos MonoinsaturadosRESUMEN
Hemp-sprouts are emerging as a new class of attractive functional food due to their numerous health benefits when compared to other sprout species. Indeed, the high content of beneficial components including polyphenols and flavonoids makes this type of food a promising and successful market. However, the available literature on this topic is limited and often conflicting as regards to the content of phytocannabinoids. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was applied in an untargeted metabolomics fashion to extracts of hemp seeds, sprouts and microgreens of nine different genotypes. Both unsupervised and supervised multivariate statistical analysis was performed to reveal variety-specific profiles of phytocannabinoids with surprisingly remarkable levels of phytocannabinoids even in chemotype V samples. Furthermore, a targeted HPLC-HRMS analysis was carried out for the quantitative determination of the major phytocannabinoids including CBDA, CBD, CBGA, CBG, CBCA, CBC, THCA, and trans-Δ9-THC. The last part of the study was focused on the evaluation of the enantiomeric composition of CBCA in hemp seeds, sprouts and microgreens in the different varieties by HPLC-CD (HPLC with online circular dichroism). Chiral analysis of CBCA showed a wide variability of its enantiomeric composition in the different varieties, thus contributing to the understanding of the intriguing stereochemical behavior of this compound in an early growth stage. However, further investigation is needed to determine the genetic factors responsible for the low enantiopurity of this compound.
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Cannabis , Semillas , Cannabis/química , Cannabis/crecimiento & desarrollo , Semillas/química , Cromatografía Líquida de Alta Presión/métodos , Cannabinoides/análisis , Cannabinoides/química , Extractos Vegetales/química , Extractos Vegetales/análisis , Espectrometría de Masas/métodos , Metabolómica/métodos , Estereoisomerismo , Dicroismo Circular/métodosRESUMEN
Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMeds) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based one, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies.
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Ácidos Grasos Monoinsaturados , Glioblastoma , Liposomas , Compuestos de Amonio Cuaternario , Ratones , Animales , Humanos , Liposomas/química , Polímeros/química , ARN Interferente Pequeño , Glioblastoma/genética , Glioblastoma/terapia , Células 3T3 NIH , Nanomedicina , Lípidos/químicaRESUMEN
[This corrects the article DOI: 10.1016/j.ijpx.2022.100136.].
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Curcumin is a well-known antioxidant used as traditional medicine in China and India since ages to treat variety of inflammatory ailments as a food supplement. Curcumin has antitumor properties with neuroprotective effects in Alzheimer's disease. Curcumin elevates brain-derived neurotrophic factor (BDNF) and dopamine (DA) levels in the brain indicating its role in substance abuse. Methamphetamine (METH) is one of the most abused substances in the world that induces profound neurotoxicity by inducing breakdown of the blood-brain barrier (BBB), vasogenic edema and cellular injuries. However, influence of curcumin on METH-induced neurotoxicity is still not well investigated. In this investigation, METH neurotoxicity and neuroprotective effects of curcumin nanodelivery were examined in a rat model. METH (20 mg/kg, i.p.) neurotoxicity is evident 4 h after its administration exhibiting breakdown of BBB to Evans blue albumin in the cerebral cortex, hippocampus, cerebellum, thalamus and hypothalamus associated with vasogenic brain edema as seen measured using water content in all these regions. Nissl attaining exhibited profound neuronal injuries in the regions of BBB damage. Normal curcumin (50 mg/kg, i.v.) 30 min after METH administration was able to reduce BBB breakdown and brain edema partially in some of the above brain regions. However, TiO2 nanowired delivery of curcumin (25 mg/kg, i.v.) significantly attenuated brain edema, neuronal injuries and the BBB leakage in all the brain areas. BDNF level showed a significant higher level in METH-treated rats as compared to saline-treated METH group. Significantly enhanced DA levels in METH-treated rats were also observed with nanowired delivery of curcumin. Normal curcumin was able to slightly elevate DA and BDNF levels in the selected brain regions. Taken together, our observations are the first to show that nanodelivery of curcumin induces superior neuroprotection in METH neurotoxicity probable by enhancing BDNF and DA levels in the brain, not reported earlier.
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Edema Encefálico , Curcumina , Metanfetamina , Fármacos Neuroprotectores , Animales , Ratas , Factor Neurotrófico Derivado del Encéfalo , Dopamina , Metanfetamina/toxicidad , Fármacos Neuroprotectores/farmacología , Nanocables/química , Sistema de Administración de Fármacos con Nanopartículas/química , Sistema de Administración de Fármacos con Nanopartículas/farmacologíaRESUMEN
Cannabis is a multifaceted plant with numerous therapeutic properties on one hand, and controversial psychotropic activities on the other hand, which are modulated by CB1 endocannabinoid receptors. Δ9-Tetrahydrocannabinol (Δ9-THC) has been identified as the main component responsible for the psychotropic effects, while its constitutional isomer cannabidiol (CBD) has shown completely different pharmacological properties. Due to its reported beneficial effects, Cannabis has gained global popularity and is openly sold in shops and online. To circumvent legal restrictions, semi-synthetic derivatives of CBD are now frequently added to cannabis products, producing "high" effects similar to those induced by Δ9-THC. The first semi-synthetic cannabinoid to appear in the EU was obtained through cyclization and hydrogenation of CBD, and is known as hexahydrocannabinol (HHC). Currently, there is limited knowledge regarding HHC, its pharmacological properties, and its prevalence, as it is not commonly investigated in routine toxicological assays. In this study, synthetic strategies were explored to obtain an excess of the active epimer of HHC. Furthermore, the two epimers were purified and individually tested for their cannabinomimetic activity. Lastly, a simple and rapid chromatographic method employing a UV detector and a high-resolution mass spectrometer was applied to identify and quantify up to ten major phytocannabinoids, as well as the HHC epimers, in commercial cannabis samples.
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Cannabidiol , Cannabinoides , Cannabis , Alucinógenos , Dronabinol/farmacología , Psicotrópicos/farmacología , Cannabinoides/farmacología , Cannabis/química , Cannabidiol/farmacología , Cannabidiol/químicaRESUMEN
Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors.
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Enfermedad de Huntington , Ratones , Animales , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/patología , Encéfalo/patología , Colesterol , Cuerpo Estriado/patología , Cognición , Modelos Animales de Enfermedad , Ratones TransgénicosRESUMEN
Spinal cord injury (SCI) is characterized by a cascade of events that lead to sensory and motor disabilities. To date, this condition is irreversible, and no cure exists. To improve myelin repair and limit secondary degeneration, we developed a multitherapy based on nanomedicines (NMeds) loaded with the promyelinating agent triiodothyronine (T3), used in combination with systemic ibuprofen and mouse nerve growth factor (mNGF). Poly-L-lactic-co-glycolic acid (PLGA) NMeds were optimized and loaded with T3 to promote sustained release. In vitro experiments confirmed the efficacy of T3-NMeds to differentiate oligodendrocyte precursor cells. In vivo rat experiments were performed in contusion SCI to explore the NMed biodistribution and efficacy of combo drugs at short- and long-term post-lesion. A strong anti-inflammatory effect was observed in the short term with a reduction of type M1 microglia and glutamate levels, but with a subsequent increase of TREM2. In the long term, an improvement of myelination in NG2-IR, an increase in MBP content, and a reduction of the demyelination area were observed. These data demonstrated that NMeds can successfully be used to obtain more controlled local drug delivery and that this multiple treatment could be effective in improving the outcome of SCIs.
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Remielinización , Traumatismos de la Médula Espinal , Ratas , Ratones , Animales , Remielinización/fisiología , Distribución Tisular , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Vaina de Mielina/patología , Inflamación/tratamiento farmacológico , Inflamación/patología , Glicoproteínas de Membrana/farmacología , Receptores InmunológicosRESUMEN
Glioblastoma multiforme (GBM) is the most common malignant brain tumor, associated with low long-term survival. Nanoparticles (NPs) developed against GBM are a promising strategy to improve current therapies, by enhancing the brain delivery of active molecules and reducing off-target effects. In particular, NPs hold high potential for the targeted delivery of chemotherapeutics both across the blood-brain barrier (BBB) and specifically to GBM cell receptors, pathways, or the tumor microenvironment (TME). In this review, the most recent strategies to deliver drugs to GBM are explored. The main focus is on how surface functionalizations are essential for BBB crossing and for tumor specific targeting. We give a critical analysis of the various ligand-based approaches that have been used to target specific cancer cell receptors and the TME, or to interfere with the signaling pathways of GBM. Despite the increasing application of NPs in the clinical setting, new methods for ligand and surface characterization are needed to optimize the synthesis, as well as to predict their in vivo behavior. An expert opinion is given on the future of this research and what is still missing to create and characterize a functional NP system for improved GBM targeting.
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Neoplasias Encefálicas , Glioblastoma , Nanopartículas , Humanos , Glioblastoma/metabolismo , Ligandos , Nanopartículas/uso terapéutico , Transporte Biológico , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Barrera Hematoencefálica/metabolismo , Sistemas de Liberación de Medicamentos , Microambiente TumoralRESUMEN
Nanoparticles (NPs) are paving the way for improved treatments for difficult to treat diseases diseases; however, much is unknown about their fate in the body. One important factor is the interaction between NPs and blood proteins leading to the formation known as the "protein corona" (PC). The PC, consisting of the Hard (HC) and Soft Corona (SC), varies greatly based on the NP composition, size, and surface properties. This highlights the need for specific studies to differentiate the PC formation for each individual NP system. This work focused on comparing the HC and SC of three NPs with different matrix compositions: a) polymeric NPs based on poly(lactic-co-glycolic) acid (PLGA), b) hybrid NPs consisting of PLGA and Cholesterol, and c) lipidic NPs made only of Cholesterol. NPs were formulated and characterized for their physico-chemical characteristics and composition, and then were incubated in human plasma. In-depth purification, identification, and statistical analysis were then performed to identify the HC and SC components. Finally, similar investigations demonstrated whether the presence of a targeting ligand on the NP surface would affect the PC makeup. These results highlighted the different PC fingerprints of these NPs, which will be critical to better understand the biological influences of the PC and improve future NP designs.
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Cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA) are known to be the major phytocannabinoids in Cannabis sativa L., along with their decarboxylated derivatives cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC). The cis isomer of Δ9-THC has been recently identified, characterized and quantified in several Cannabis sativa varieties, which had been heated (decarboxylated) before the analysis. Since decarboxylation alters the original phytocannabinoids composition of the plant, this work reports the identification and characterization of the carboxylated precursor cis-Δ9-THCA. The compound was also synthesized and used as analytical standard for the development and validation of a liquid chromatography coupled to high resolution mass spectrometry-based method for its quantification in ten Cannabis sativa L. samples from different chemotypes. The highest concentrations of cis-Δ9-THCA were found in CBD-rich varieties, lower levels were observed in cannabigerol (CBG)-rich varieties (chemotype IV) and in those varieties with a balanced level of both CBD and THC (chemotype III), while its levels were not detectable in cannabichromene (CBC)-rich varieties (chemotype VI). The presence of the cis isomer of THC and THCA raises the question on whether to include or not this species in the calculation of the total amount of THC to classify a cannabis variety as a drug-type or a fiber-type (hemp).
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Cannabidiol , Cannabis , Cannabidiol/análisis , Cannabis/química , Cromatografía Líquida de Alta Presión/métodos , Dronabinol/análogos & derivados , Dronabinol/análisisRESUMEN
Glioblastoma Multiforme (GBM) is a devastating disease with a low survival rate and few efficacious treatment options. The fast growth, late diagnostics, and off-target toxicity of currently used drugs represent major barriers that need to be overcome to provide a viable cure. Nanomedicines (NMeds) offer a way to overcome these pitfalls by protecting and loading drugs, increasing blood half-life, and being targetable with specific ligands on their surface. In this study, the FDA-approved polymer poly (lactic-co-glycolic) acid was used to optimise NMeds that were surface modified with a series of potential GBM-specific ligands. The NMeds were fully characterised for their physical and chemical properties, and then in vitro testing was performed to evaluate cell uptake and GBM cell specificity. While all targeted NMeds showed improved uptake, only those decorated with the-cell surface vimentin antibody M08 showed specificity for GBM over healthy cells. Finally, the most promising targeted NMed candidate was loaded with the well-known chemotherapeutic, paclitaxel, to confirm targeting and therapeutic effects in C6 GBM cells. These results demonstrate the importance of using well-optimised NMeds targeted with novel ligands to advance delivery and pharmaceutical effects against diseased cells while minimising the risk for nearby healthy cells.
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Reactive oxygen species (ROS)-sensitive drug delivery systems (DDS) specifically responding to altered levels of ROS in the pathological microenvironment have emerged as an effective means to enhance the pharmaceutical efficacy of conventional nanomedicines, while simultaneously reducing side effects. In particular, the use of the biocompatible, biodegradable, and non-toxic ROS-responsive thioketal (TK) functional group in the design of smart DDS has grown exponentially in recent years. In the design of TK-based DDS, different technological uses of TK have been proposed to overcome the major limitations of conventional DDS counterparts including uncontrolled drug release and off-target effects. This review will focus on the different technological uses of TK-based biomaterials in smart nanomedicines by using it as a linker to connect a drug on the surface of nanoparticles, form prodrugs, as a core component of the DDS to directly control its structure, to control the opening of drug-releasing gates or to change the conformation of the nano-systems. A comprehensive view of the various uses of TK may allow researchers to exploit this reactive linker more consciously while designing nanomedicines to be more effective with improved disease-targeting ability, providing novel therapeutic opportunities in the treatment of many diseases.
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L-Kynurenine (KYN) and kynurenic acid (KYNA) are products of the metabolism of L-tryptophan (TRP) in the central nervous system of animals, but they are not commonly found in plants. In particular, KYNA is known for its interesting pharmacological properties (anti-oxidative, anti-inflammatory, hypolipidemic, and neuroprotective), which suggest a potential functional food ingredient role. The three compounds were identified in samples of Cannabis sativa L. by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry using an untargeted metabolomics approach. Their concentrations were evaluated using a targeted metabolomics method in three organs of the plant (roots, stem, and leaves) in soil at two different growth stages and in hydroponics conditions. The distribution of TRP, KYN and KYNA was found tendentially higher in leaves compared to stem and roots and changed over time. Moreover, the levels of KYNA found in this study are unprecedentedly high compared to those found so far in other plant species, suggesting that Cannabis sativa L. could be a promising alternative source of this metabolite.
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Cannabis , Quinurenina , Animales , Humanos , Ácido Quinurénico , Quinurenina/metabolismo , Neurotransmisores , Triptófano/metabolismoRESUMEN
Tunneling nanotubes (TNTs), discovered in 2004, are thin, long protrusions between cells utilized for intercellular transfer and communication. These newly discovered structures have been demonstrated to play a crucial role in homeostasis, but also in the spreading of diseases, infections, and metastases. Gaining much interest in the medical research field, TNTs have been shown to transport nanomedicines (NMeds) between cells. NMeds have been studied thanks to their advantageous features in terms of reduced toxicity of drugs, enhanced solubility, protection of the payload, prolonged release, and more interestingly, cell-targeted delivery. Nevertheless, their transfer between cells via TNTs makes their true fate unknown. If better understood, TNTs could help control NMed delivery. In fact, TNTs can represent the possibility both to improve the biodistribution of NMeds throughout a diseased tissue by increasing their formation, or to minimize their formation to block the transfer of dangerous material. To date, few studies have investigated the interaction between NMeds and TNTs. In this work, we will explain what TNTs are and how they form and then review what has been published regarding their potential use in nanomedicine research. We will highlight possible future approaches to better exploit TNT intercellular communication in the field of nanomedicine.
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Estructuras de la Membrana Celular/metabolismo , Animales , Transporte Biológico/fisiología , Humanos , Nanomedicina/métodos , Nanotubos , Distribución Tisular/fisiologíaRESUMEN
A drawback in the development of treatments that can reach the retina is the presence of barriers in the eye that restrain compounds from reaching the target. Intravitreal injections hold promise for retinal delivery, but the natural defenses in the vitreous can rapidly degrade or eliminate therapeutic molecules. Injectable hydrogel implants, which act as a reservoir, can allow for long-term drug delivery with a single injection into the eye, but still suffer due to the fast clearance of the released drugs when traversing the vitreous and random diffusion that leads to lower pharmaceutic efficacy. A combination with HA-covered nanoparticles, which can be released from the gel and more readily pass through the vitreous to increase the delivery of therapeutic agents to the retina, represents an advanced and elegant way to overcome some of the limitations in eye drug delivery. In this article, we developed hybrid PLGA-Dotap NPs that, due to their hyaluronic acid coating, can improve in vivo distribution throughout the vitreous and delivery to retinal cells. Moreover, a hydrogel implant was developed to act as a depot for the hybrid NPs to better control and slow their release. These results are a first step to improve the treatment of retinal diseases by protecting and transporting the therapeutic treatment across the vitreous and to improve treatment options by creating a depot system for long-term treatments.
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Enzymes, as natural and potentially long-term treatment options, have become one of the most sought-after pharmaceutical molecules to be delivered with nanoparticles (NPs); however, their instability during formulation often leads to underwhelming results. Various molecules, including the Tween® polysorbate series, have demonstrated enzyme activity protection but are often used uncontrolled without optimization. Here, poly(lactic-co-glycolic) acid (PLGA) NPs loaded with ß-glucosidase (ß-Glu) solutions containing Tween® 20, 60, or 80 were compared. Mixing the enzyme with Tween® pre-formulation had no effect on particle size or physical characteristics, but increased the amount of enzyme loaded. More importantly, NPs made with Tween® 20:enzyme solutions maintained significantly higher enzyme activity. Therefore, Tween® 20:enzyme solutions ranging from 60:1 to 2419:1 mol:mol were further analyzed. Isothermal titration calorimetry analysis demonstrated low affinity and unquantifiable binding between Tween® 20 and ß-Glu. Incorporating these solutions in NPs showed no effect on size, zeta potential, or morphology. The amount of enzyme and Tween® 20 in the NPs was constant for all samples, but a trend towards higher activity with higher molar rapports of Tween® 20:ß-Glu was observed. Finally, a burst release from NPs in the first hour with Tween®:ß-Glu solutions was the same as free enzyme, but the enzyme remained active longer in solution. These results highlight the importance of stabilizers during NP formulation and how optimizing their use to stabilize an enzyme can help researchers design more efficient and effective enzyme loaded NPs.