Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing

Clinical validation of risk scoring systems to predict risk of delayed bleeding after EMR of large colorectal lesions

[Background and Aims]: The Endoscopic Resection Group of the Spanish Society of Endoscopy (GSEED-RE) model and the Australian Colonic Endoscopic Resection (ACER) model were proposed to predict delayed bleeding (DB) after EMR of large superficial colorectal lesions, but neither has been validated. We validated and updated these models.[Methods]: A multicenter cohort study was performed in patients with nonpedunculated lesions ≥20 mm removed by EMR. We assessed the discrimination and calibration of the GSEED-RE and ACER models. Difficulty performing EMR was subjectively categorized as low, medium, or high. We created a new model, including factors associated with DB in 3 cohort studies.[Results]: DB occurred in 45 of 1034 EMRs (4.5%); it was associated with proximal location (odds ratio [OR], 2.84; 95% confidence interval [CI], 1.31-6.16), antiplatelet agents (OR, 2.51; 95% CI, .99-6.34) or anticoagulants (OR, 4.54; 95% CI, 2.14-9.63), difficulty of EMR (OR, 3.23; 95% CI, 1.41-7.40), and comorbidity (OR, 2.11; 95% CI, .99-4.47). The GSEED-RE and ACER models did not accurately predict DB. Re-estimation and recalibration yielded acceptable results (GSEED-RE area under the curve [AUC], .64 [95% CI, .54-.74]; ACER AUC, .65 [95% CI, .57-.73]). We used lesion size, proximal location, comorbidity, and antiplatelet or anticoagulant therapy to generate a new model, the GSEED-RE2, which achieved higher AUC values (.69-.73; 95% CI, .59-.80) and exhibited lower susceptibility to changes among datasets.[Conclusions]: The updated GSEED-RE and ACER models achieved acceptable prediction levels of DB. The GSEED-RE2 model may achieve better prediction results and could be used to guide the management of patients after validation by other external groups. (Clinical trial registration number: NCT 03050333.)Research support for this study was received from “La Caixa/Caja Navarra” Foundation (ID 100010434;project PR15/11100006)

Liposomal formulations for treating lysosomal storage disorders

Lysosomal storage disorders (LSD) are a group of rare life-threatening diseases caused by a lysosomal dysfunction, usually due to the lack of a single enzyme required for the metabolism of macromolecules, which leads to a lysosomal accumulation of specific substrates, resulting in severe disease manifestations and early death. There is currently no definitive cure for LSD, and despite the approval of certain therapies, their effectiveness is limited. Therefore, an appropriate nanocarrier could help improve the efficacy of some of these therapies. Liposomes show excellent properties as drug carriers, because they can entrap active therapeutic compounds offering protection, biocompatibility, and selectivity. Here, we discuss the potential of liposomes for LSD treatment and conduct a detailed analysis of promising liposomal formulations still in the preclinical development stage from various perspectives, including treatment strategy, manufacturing, characterization, and future directions for implementing liposomal formulations for LSD.This work has received funding from: Grant Severo Ochoa CEX2019-000917-S funded by Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación MCIN/AEI/10.13039/501100011033; Project MOL4BIO PID2019-105622RB-I00 funded by Agencia Estatal de Investigación - Ministerio de Ciencia e Innovación MCIN/AEI/10.13039/501100011033; Project PHOENIX-OITB funded by European Union’s Horizon 2020 research and innovation programme under grant agreement No 953110; Project SMART4FABRY funded by European Union’s Horizon 2020 research and innovation programme under grant agreement No 720942. The authors are also grateful to the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Liposomes et son utilisation pour l'administration d'enzymes

The present invention refers to a liposome comprising: a) a phospholipid; b) cholesterol (chol); c) a conjugate comprising a cholesterol moiety, a polyethylene glycol (PEG) moiety and a peptide moiety comprising a RGD sequence, wherein the PEG moiety is covalently attached to the cholesterol moiety by one end via a bond of the type alkyl ether and is covalently attached to the peptide moiety comprising the RGD sequence by the other end: d) a non-lipid cationic surfactant present in a percentage of less than 30% mol in respect to the total mol of the components of the liposome a), b), c) and d); and e) alpha-galactosidase (GLA) enzyme present in a ratio of micrograms of GLA in respect to the total milligrams of the components of the liposome a), b), c) and d) of between and including 2 to 35. It also refers to a pharmaceutical composition that comprises it and to the liposome or the pharmaceutical composition for use as a medicament, in particular for use in the treatment of Fabry disease. It also refers to a process for the production of the liposome.NoConsejo Superior de Investigaciones Científicas (CSIC), Consorcio Centro de Investigación Biomédica En Red, Fundació Hospital Universitari Vall D'Hebron - Institut de Recerca, Nanomol Technologies, S.L., LeanbioA1 Solicitud de patente con informe sobre el estado de la técnic

Liposomes et leur utilisation pour l'administration d'enzymes

[EN] The present invention refers to a liposome comprising: a) a phospholipid; b) cholesterol (chol); c) a conjugate comprising a cholesterol moiety, a polyethylene glycol (PEG) moiety and a peptide moiety comprising a RGD sequence, wherein the PEG moiety is covalently attached to the cholesterol moiety by one end via a bond of the type alkyl ether and is covalently attached to the peptide moiety comprising the RGD sequence by the other end: d) a non-lipid cationic surfactant present in a percentage of less than 30% mol in respect to the total mol of the components of the liposome a), b), c) and d); and e) alpha-galactosidase (GLA) enzyme present in a ratio of micrograms of GLA in respect to the total milligrams of the components of the liposome a), b), c) and d) of between and including 2 to 35. It also refers to a pharmaceutical composition that comprises it and to the liposome or the pharmaceutical composition for use as a medicament, in particular for use in the treatment of Fabry disease. It also refers to a process for the production of the liposome.[FR] La présente invention concerne un liposome comprenant : a) un phospholipide ; b) cholestérol (chol) ; c) un conjugué comprenant une fraction de cholestérol, une fraction de polyéthylène glycol (PEG) et une fraction peptidique comprenant une séquence RGD, la fraction PEG étant liée de manière covalente à la fraction de cholestérol par une extrémité par l'intermédiaire d'une liaison du type éther alcoylique et étant liée de manière covalente à la fraction peptidique comprenant la séquence RGD par l'autre extrémité : d) un agent tensioactif cationique non lipidique présent dans un pourcentage inférieur à 30% molaire par rapport au total molaire des composants du liposome a), b), c) et d) ; et e) l'enzyme alpha-galactosidase (GLA) présente dans un rapport de microgrammes de GLA par rapport aux milligrammes totaux des composants du liposome a), b), c) et d) de 2 à 35 inclus. L'invention concerne également une composition pharmaceutique qui le comprend et le liposome ou la composition pharmaceutique pour une utilisation comme médicament, en particulier pour une utilisation dans le traitement de la maladie de Fabry. L'invention concerne également un procédé de production du liposome.NoConsejo Superior de Investigaciones Científicas (CSIC), Consorcio Centro de Investigación Biomédica En Red, Fundació Hospital Universitari Vall D'Hebron - Institut de Recerca, Nanomol Technologies, S.L., LeanbioA1 Solicitud de patente con informe sobre el estado de la técnic

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.The authors acknowledge the financial support from European Commission through H2020 program of the Smart-4-Fabry project (ID 720942). We acknowledge financial support to our research from Instituto de Salud Carlos III, through “Acciones CIBER”. The Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN) is 2008–2011, financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Authors acknowledge financial support from the Agencia Estatal de Investigación-Ministerio de Ciencia e Innovación through the “Severo Ochoa” Programme for Centres of Excellence in R & D (CEX2019-000917-S). This work was also financed by 1084 the Ministerio de Ciencia e Innovación (PID2019-105622RB-1085 I00). We also thank the denomination of the 1101 consolidated group from Generalitat de Catalunya: 2017-1102 SGR-1439 (M.R.) and 2017-SGR-918 (J.V.). We also acknowledge the ICTS “NANBIOSIS”, more specifically the support from the Protein Production Platform of CIBER-BBN/IBB, at the UAB SepBioES scientific-technical service (www.nanbiosis.es/unit/u1-proteinproduction-platform-ppp/), the Soft Materials Service linked to Biomaterial Processing and Nanostructuring Unit at ICMAB-CSIC (www.nanbiosis.es/portfolio/u6-biomaterial-processing-and-nanostructuring-unit/) and the Peptide Synthesis unit at the IQAC-CSIC (www.nanbiosis.es/portfolio/u3-synthesis-of-peptides-unit/). N. A-G. is supported by a PERIS grant from the Catalan Government (SLT006/17/270). J. T-M. is supported by a FI-AGAUR grant from the Catalan Government and the European Social Fund (ESF-Investing in your future) of the European Union. This work has been done in the framework of the JT-M. doctorate in Materials Science of the Universitat Autònoma de Barcelona. The technical assistance of Inbal Ionita, Ella Kesselman and Mingming Zhang from Technion - Israel Institute of Technology; and Ramon González from ICMAB-CSIC is acknowledged.Peer reviewe

Recombinant Human Epidermal Growth Factor/Quatsome Nanoconjugates: A Robust Topical Delivery System for Complex Wound Healing

A multitude of microparticles and nanoparticles is developed to improve the delivery of different small drugs and large biomolecules, which are subject to several hindering biological barriers that limit their optimal biodistribution and therapeutic effects. Here, a soft, reliable, and scalable method based on compressed CO2 is reported for obtaining nanoconjugates of recombinant human epidermal growth factor and nanovesicles called quatsomes, where the latter consists of cholesterol and cetyltrimethylammonium bromide. These nanoconjugates exhibit appropriate values of the major critical quality attributes of colloidal nanomedicines, such as controlled and narrow nanoscopic particle size distribution (which play important roles in determining their stability), drug loading, drug release, drug protection, targeting ability, and bioactivity. Also, they exhibit a dual action by 1) inbuilt antimicrobial activity preventing infections and 2) promoting regeneration of granulation tissue and re‐epithelialization with complete closure of complex wounds. Therefore, such nanoconjugates are a potential nanomedicine for the topical treatment of complex wounds, particularly diabetic foot ulcers and venous leg ulcers.L.F.‐T and H.S. contributed equally to this work. This work was funded by the Spanish Ministry/FEDER project MOTHER MAT2016–80826‐R; the Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine (CIBER‐BBN); the Generalitat de Catalunya (2017SGR918); and the Spanish Ministry of Economy and Competitiveness by the Severo Ochoa FUNFUTURE (CEX2019‐000917‐S) Excellence Centre distinction. This work was also co‐financed by the European Union through FEDER project NANONAFRES (COMRDI15‐1‐0023). The authors thank Milagros Font and Yenay Diaz from CIGB for technical assistance. The authors also thank the Industrial Doctorates Plan of Agaur‐Generalitat de Catalunya (2018 DI 057). Some figures of this work were created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License.Peer reviewe

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.info:eu-repo/semantics/publishedVersio

Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems—quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.This work was supported by the financial support from European Commission through the H2020 program (Smart-4-Fabry project, ID 720942). The Networking Research Centre on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN) is financed by the Instituto de Salud Carlos III (ISCIII) with assistance from the European Regional Development Fund (ERDF). This work was also financed by the Ministerio de Ciencia e Innovación (PID2019-105622RB-I00). The work was also partially funded by ISCIII (PI18_00871 cofounded by ERDF) and CIBER-BBN (EXPLORE) granted to I.A. We also acknowledge the ICTS “NANBIOSIS”, more specifically, the support from the Protein Production Platform of CIBER-BBN/IBB, at the UAB SepBioES scientific-technical service (https://www.nanbiosis.es/portfolio/u1-protein-production-platform-ppp/), the Soft Materials Service linked to Biomaterial Processing and Nanostructuring Unit (U6) at ICMAB-CSIC (www.nanbiosis.es/portfolio/u6-biomaterial-processing-and-nanostructuring-unit/), the Synthesis of Peptide Unit (U3) at the IQAC-CSIC (www.nanbiosis.es/portfolio/u3-synthesis-of-peptides-unit/), and the In Vivo Experimental Platform of the Functional Validation & Preclinical Research (FVPR) area (http://www.nanbiosis.es/portfolio/u20-in-vivo-experimental-platform/). We also thank the denomination of the consolidated group from Generalitat de Catalunya: 2017-SGR-1439 (M.R.) and 2017-SGR-918 (J.V.). J.T.-M. thanks the financial support by the FI-AGAUR grant by the Generalitat de Catalunya, especially the Secretary of Universities and Research of the Department of Business and Knowledge of the Generalitat de Catalunya and the European Social Fund (ESF–Investing in your future) of the European Union. This work has been done in the framework of the J.T.-M. doctorate in Materials Science of the Universitat Autònoma de Barcelona. N.G.-A. is supported by a PERIS grant from the Catalan Government (SLT006/17/270). Authors acknowledge financial support from the Agencia Estatal de Investigación-Ministerio de Ciencia e Innovación through the “Severo Ochoa” Programme for Centres of Excellence in R&D (CEX2019-000917-S).Peer reviewe