1840856612bae0cb780150cf5cb66ada pmc_9322090.pdf e7b1ef48231f79dfa0f04317546e61dd99d3ea92 pmc_9322090.pdf d352662b6e8836a328bc27b5f6858a5b2db8093daf07bd72b427f9e41146162e pmc_9322090.pdf Title: Fluorescent PLGA Nanocarriers for Pulmonary Administration: Influence of the Surface Charge Subject: Nearly four million yearly deaths can be attributed to respiratory diseases, prompting a huge worldwide health emergency. Additionally, the COVID-19 pandemic’s death toll has surpassed six million, significantly increasing respiratory disease morbidity and mortality rates. Despite recent advances, it is still challenging for many drugs to be homogeneously distributed throughout the lungs, and specifically to reach the lower respiratory tract with an accurate sustained dose and minimal systemic side effects. Engineered nanocarriers can provide increased therapeutic efficacy while lessening potential biochemical adverse reactions. Poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer, has attracted significant interest as an inhalable drug delivery system. However, the influence of the nanocarrier surface charge and its intratracheal instillation has not been addressed so far. In this study, we fabricated red fluorescent PLGA nanocapsules (NCs)—Cy5/PLGA—with either positive (Cy5/PLGA+) or negative surface charge (Cy5/PLGA-). We report here on their excellent colloidal stability in culture and biological media, and after cryo-storage. Their lack of cytotoxicity in two relevant lung cell types, even for concentrations as high as 10 mg/mL, is also reported. More importantly, differences in the NCs’ cell uptake rates and internalization capacity were identified. The uptake of the anionic system was faster and in much higher amounts—10-fold and 2.5-fold in macrophages and epithelial alveolar cells, respectively. The in vivo study demonstrated that anionic PLGA NCs were retained in all lung lobules after 1 h of being intratracheally instilled, and were found to accumulate in lung macrophages after 24 h, making those nanocarriers especially suitable as a pulmonary immunomodulatory delivery system with a marked translational character. Keywords: nanocarriers; lung; PLGA; surface charge; intratracheal instillation; cell uptake Author: Aina Areny-Balagueró, Wid Mekseriwattana, Marta Camprubí-Rimblas, Andrea Stephany, Ariana Roldan, Anna Solé-Porta, Antonio Artigas, Daniel Closa and Anna Roig Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Jul 11 16:11:22 2022 CEST ModDate: Mon Jul 11 16:17:07 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5152003 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- XWOCXD+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 QXCFRE+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 JJRZKG+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 RNFFOO+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 YHLBEF+VnURWPalladioL Type 1 Custom yes yes no 28 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TrueType WinAnsi yes yes no 290 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-06-17 02:31:38 CEST RepresentationInformation: pmc_9322090.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-09-05 13:33:02 CEST Size: 5152003 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 464 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Materials Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Suppliers and Purity of the Chemical Reagents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Synthesis of Amine-Functionalized PLGA (PLGA-NH2) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Synthesis of Fluorescent PLGA (PLGA-Cy5) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Formulation of Fluorescent PLGA NCs with Different Surface Charges Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Characterization of PLGA NCs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Cell Harvesting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: In Vitro MTS Cytotoxicity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Confocal Analysis for Cy5/PLGA- and Cy5/PLGA+ Cellular Uptake In Vitro Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Animals Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: In Vivo Lung Biodistribution and Cellular Uptake of NCs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: Synthesis of PLGA Nanocapsules Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Colloidal Stability of the PLGA Nanocapsules in Culture and Biological Media Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Nanocapsules’ Cytotoxicity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Nanocapsules’ Cellular Uptake Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Nanocapsules’ Biodistribution and Cellular Uptake In Vivo Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Info: Title: Fluorescent PLGA Nanocarriers for Pulmonary Administration: Influence of the Surface Charge Author: Aina Areny-Balagueró, Wid Mekseriwattana, Marta Camprubí-Rimblas, Andrea Stephany, Ariana Roldan, Anna Solé-Porta, Antonio Artigas, Daniel Closa and Anna Roig Subject: Nearly four million yearly deaths can be attributed to respiratory diseases, prompting a huge worldwide health emergency. Additionally, the COVID-19 pandemic’s death toll has surpassed six million, significantly increasing respiratory disease morbidity and mortality rates. Despite recent advances, it is still challenging for many drugs to be homogeneously distributed throughout the lungs, and specifically to reach the lower respiratory tract with an accurate sustained dose and minimal systemic side effects. Engineered nanocarriers can provide increased therapeutic efficacy while lessening potential biochemical adverse reactions. Poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer, has attracted significant interest as an inhalable drug delivery system. However, the influence of the nanocarrier surface charge and its intratracheal instillation has not been addressed so far. In this study, we fabricated red fluorescent PLGA nanocapsules (NCs) Cy5/PLGA with either positive (Cy5/PLGA+) or negative surface charge (Cy5/PLGA-). We report here on their excellent colloidal stability in culture and biological media, and after cryo-storage. Their lack of cytotoxicity in two relevant lung cell types, even for concentrations as high as 10 mg/mL, is also reported. More importantly, differences in the NCs’ cell uptake rates and internalization capacity were identified. The uptake of the anionic system was faster and in much higher amounts 10-fold and 2.5-fold in macrophages and epithelial alveolar cells, respectively. The in vivo study demonstrated that anionic PLGA NCs were retained in all lung lobules after 1 h of being intratracheally instilled, and were found to accumulate in lung macrophages after 24 h, making those nanocarriers especially suitable as a pulmonary immunomodulatory delivery system with a marked translational character. 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