Biodegradable nanoparticles sequentially decorated with Polyethyleneimine and Hyaluronan for the targeted delivery of docetaxel to airway cancer cells.
ABSTRACT: Novel polymeric nanoparticles (NPs) specifically designed for delivering chemotherapeutics in the body and aimed at improving treatment activity and selectivity, cover a very relevant area in the field of nanomedicine. Here, we describe how to build a polymer shell of Hyaluronan (HA) and Polyethyleneimine (PEI) on biodegradable NPs of poly(lactic-co-glycolic) acid (PLGA) through electrostatic interactions and to achieve NPs with unique features of sustained delivery of a docetaxel (DTX) drug cargo as well as improved intracellular uptake.A stable PEI or HA/PEI shell could be obtained by careful selection of layering conditions. NPs with exquisite stability in salt and protein-rich media, with size and surface charge matching biological requirements for intravenous injection and endowed with sustained DTX release could be obtained. Cytotoxicity, uptake and activity of both PLGA/PEI/HA and PLGA/PEI NPs were evaluated in CD44(+) (A549) and CD44(-) (Calu-3) lung cancer cells. In fact, PEI-coated NPs can be formed after degradation/dissociation of the surface HA because of the excess hyaluronidases overexpressed in tumour interstitium. There was no statistically significant cytotoxic effect of PLGA/PEI/HA and PLGA/PEI NPs in both cell lines, thus suggesting that introduction of PEI in NP shell was not hampered by its intrinsic toxicity. Intracellular trafficking of NPs fluorescently labeled with Rhodamine (RHO) (RHO-PLGA/PEI/HA and RHO-PLGA/PEI NPs) demonstrated an increased time-dependent uptake only for RHO-PLGA/PEI/HA NPs in A549 cells as compared to Calu-3 cells. As expected, RHO-PLGA/PEI NP uptake in A549 cells was comparable to that observed in Calu-3 cells. RHO-PLGA/PEI/HA NPs internalized into A549 cells showed a preferential perinuclear localization. Cytotoxicity data in A549 cells suggested that DTX delivered through PLGA/PEI/HA NPs exerted a more potent antiproliferative activity than free DTX. Furthermore, DTX-PLGA/PEI NPs, as hypothetical result of hyaluronidase-mediated degradation in tumor interstitium, were still able to improve the cytotoxic activity of free DTX.Taken together, results lead us to hypothesize that biodegradable NPs coated with a PEI/HA shell represent a very promising system to treat CD44 overexpressing lung cancer. In principle, this novel nanocarrier can be extended to different single drugs and drug combinations taking advantage of the shell and core properties.
Project description:The presence of rare but highly tumorigenic cancer stem cells (CSCs) within the tumors is recognized as one of the major reasons of failure of conventional chemotherapies, mainly attributed to the development of drug resistance and increasing metastatic potential. Here, we propose a therapeutic strategy based on the simultaneous delivery of docetaxel (DTX) and the photosensitizer meso-tetraphenyl chlorine disulfonate (TPCS2a) using hyaluronic acid (HA) coated polymeric nanoparticles (HA-NPs) for the targeting and killing of CD44 over-expressing breast cancer (BC) cells, both differentiated and CSCs (CD44high/CD24low population), thus combining chemotherapy and photodynamic therapy (PDT). Using the CD44high MDA-MB-231 and the CD44low MCF-7 cells, we demonstrated the occurrence of CD44-mediated uptake of HA-NPs both in monolayers and mammosphere cultures enriched in CSCs. Cell treatments showed that combination therapy using co-loaded NPs (HA@DTX/TPCS2a-NPs) had superior efficacy over monotherapies (HA@DTX-NPs or HA@TPCS2a-NPs) in reducing the self-renewal capacity, measured as mammosphere formation efficiency, and in eradicating the CSC population evaluated with aldehyde dehydrogenase activity assay and CD44/CD24 immunostaining. In summary, these in vitro studies demonstrated for the first time the potential of the combination of DTX-chemotherapy and TPCS2a-PDT for killing CSCs using properly designed NPs.
Project description:The purpose of this study was to modulate macrophage polarity from the pro-inflammatory M1 to anti-inflammatory M2 phenotype using plasmid DNA (pDNA) expressing interleukin-4 (IL4) or interleukin-10 (IL10)-encapsulated in hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles (NPs). The HA-PEI/pDNA NPs with spherical shape, average size of 186 nm were efficiently internalized by J774A.1 macrophages. Transfection of HA-PEI/pDNA-IL4 and HA-PEI/pDNA-IL10 NPs increased IL4 and IL10 gene expression in J774 macrophages which could re-program the macrophages from M1 to M2 phenotype as evidenced by a significant increase in the Arg/iNOS level, and upregulation of CD206 and CD163 compared to untreated macrophages. Following intraperitoneal (IP) injection to C57BL/6 mice, HA-PEI NPs effectively targeted peritoneal macrophages over-expressing CD44 receptor. In an in vivo model of stimulated peritoneal macrophages, IP administration of HA-PEI/pDNA-IL4 and HA-PEI/pDNA-IL10 to C57BL/6 mice significantly increased the Arg/iNOS ratio and CD163 expression in the cells. Furthermore, HA-PEI/pDNA-IL10 NPs significantly increased peritoneal and serum IL10 levels which effectively suppressed LPS-induced inflammation by reducing level of TNF-? and IL-1? in peritoneal macrophages and in the peritoneal fluid. The results demonstrated that pDNA-IL10-encapsulate HA-PEI NPs skewed macrophage functional polarity from M1 toward an anti-inflammatory M2 phenotype which may be a promising platform for the treatment of inflammatory diseases.
Project description:The aim of this study was to evaluate macrophages repolarization from pro-inflammatory M1 to anti-inflammatory M2 phenotype upon transfection with microRNA-223 (miR-223) duplexes and miR-223 expressing plasmid DNA encapsulated in CD44-targeting hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles (NPs). The HA-PEI/miR-223 NPs with spherical shape and an average diameter of 200 nm were efficiently internalized by J774A.1 alveolar and primary peritoneal macrophages and non-cytotoxic at HA-PEI concentration less than 200 ?g/mL. Transfection of HA-PEI/miR-223 NPs in J774A.1 macrophages showed significantly higher miR-223 expression than that with HA-PEI/plasmid DNA expressing miR-223 (pDNA-miR-223). HA-PEI/miR-223 NPs mediated transfection increased miR-223 expression to 90 fold in primary peritoneal macrophages compared to untreated cells. The overexpression of miR-223 in both J774A.1 and peritoneal macrophages induced a phenotypic change from M1 to M2 state as indicated by a decrease in iNOS-2 (M1 marker) and an increase in Arg-1 (M2 marker) levels compared to those in lipopolysaccharide (LPS) and interferon-gamma (IFN-?)-stimulated macrophages (M1). The change in macrophage phenotype by HA-PEI/miR-223 NPs could suppress the inflammation in peritoneal macrophages induced by LPS as evidenced by a significant decrease in pro-inflammatory cytokine levels TNF-?, IL-1? and IL-6, compared to LPS-stimulated peritoneal macrophages without treatment. The results demonstrated that miR-223-encapsulated HA-PEI NPs modulated macrophage polarity toward an anti-inflammatory M2 phenotype, which has potential for the treatment of inflammatory diseases.
Project description:BACKGROUND: Effective cancer chemotherapy remains an important issue in cancer treatment, and signal transducer and activator of transcription-3 (Stat3) activation leads to cellular resistance of anticancer agents. Polymers are ideal vectors to carry both chemotherapeutics and small interfering ribonucleic acid (siRNA) to enhance antitumor efficacy. In this paper, poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with paclitaxel and Stat3 siRNA were successfully synthesized, and their applications in cancer cells were investigated. METHODS: Firstly, paclitaxel was enclosed by PLGA nanoparticles through solvent evaporation. They were then coated with cationic polyethylenimine polymer (PLGA-PEI-TAX), enabling it to carry Stat3 siRNA on its surface through electrostatic interactions (PLGA-PEI-TAX-S3SI). The size, zeta potential, deliver efficacy, and release profile of the PLGA nanocomplexes were characterized in vitro. The cellular uptake, intracellular nanoparticle trajectory, and subsequent cellular events were evaluated after treatment with various PLGA nanocomplexes in human lung cancer A549 cells and A549-derived paclitaxel-resistant A549/T12 cell lines with ?-tubulin mutation. RESULTS: A549 and A549/T12 cells contain constitutively activated Stat3, and silencing Stat3 by siRNA made both cancer cells more sensitive to paclitaxel. Therefore, PLGA-PEI-TAX-S3SI was synthesized to test its therapeutic role in A549 and A549/T12 cells. Transmission electron microscopy showed the size of PLGA-PEI-TAX-S3SI to be around 250 nm. PLGA-PEI nanoparticles were nontoxic. PLGA-PEI-TAX was taken up by A549 and A549/T12 cells more than free paclitaxel, and they induced more condensed microtubule bundles and had higher cytotoxicity in these cancer cells. Moreover, the yellowish fluorescence observed in the cytoplasm of the cancer cells indicates that the PLGA-PEI nanoparticles were still simultaneously delivering Oregon Green paclitaxel and cyanine-5-labeled Stat3 siRNA 3 hours after treatment. Furthermore, after the cancer cells were incubated with the synthesized PLGA nanocomplexes, PLGA-PEI-TAX-S3SI suppressed Stat3 expression and induced more cellular apoptosis in A549 and A549/T12 cells compared with PLGA-PEI-TAX. CONCLUSION: The PLGA-PEI-TAX-S3SI complex provides a new therapeutic strategy to control cancer cell growth.
Project description:Effective induction of an antigen-specific cytotoxic T lymphocyte (CTL) immune response is one of the key goals of cancer immunotherapy. We report the design and fabrication of polyethylenimine (PEI)-coated polymer nanoparticles (NPs) as efficient antigen-delivery carriers that can induce antigen cross-presentation and a strong CTL response. After synthesis of poly(d,l-lactide-co-glycolide) (PLGA) NPs containing ovalbumin (OVA) by the double-emulsion solvent-evaporation method, cationic-charged PLGA NPs were generated by coating them with PEI. In a methyl tetrazolium salt assay, no discernible cytotoxic effect of PEI-coated PLGA (OVA) NPs was observed. The capacity and mechanism of PEI-coated PLGA (OVA) NPs for antigen delivery and cross-presentation on dendritic cells (DCs) were determined by fluorescence microscopy and flow cytometry. PEI-coated PLGA (OVA) NPs were internalized efficiently via phagocytosis or macropinocytosis in DCs and induced efficient cross-presentation of the antigen on MHC class I molecules via both endosome escape and a lysosomal processing mechanism. The DCs treated with PEI-coated PLGA (OVA) NPs induced a release of IL-2 cytokine from OVA-specific CD8-OVA1.3 T cells more efficiently than DCs treated with PLGA (OVA) NPs. Therefore, the PEI-coated PLGA (OVA) NPs can induce antigen cross-presentation and are expected to be used for induction of a strong CTL immune response and for efficient anticancer immunotherapy.
Project description:Recently, photothermal therapy has become a promising strategy in tumor treatment. However, the therapeutic effect was seriously hampered by the low tissue penetration of laser. Therefore, in this study, radiofrequency (RF) with better tissue penetration was used for tumor hyperthermia. First, one type of gold nanorods (AuNRs) suitable for RF hyperthermia was selected. Then, poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with AuNRs and docetaxel (DTX) (PLGA/AuNR/DTX) NPs were constructed. Finally, manganese dioxide (MnO2) ultrathin nanofilms were coated on the surfaces of PLGA/AuNR/DTX NPs by the reduction of KMnO4 to construct the PLGA/AuNR/DTX@MnO2 drug delivery system. This drug delivery system can not only be used for the combined therapy of chemotherapy and RF hyperthermia but can also produce Mn2+ to enable magnetic resonance imaging. Furthermore, the RF hyperthermia and the degradation of MnO2 can significantly promote the controlled drug release in a tumor region. The in vitro and in vivo results suggested that the PLGA/AuNR/DTX@MnO2 multifunctional drug delivery system is a promising nanoplatform for effective cancer theranostic applications.
Project description:Approaches for the synthesis of biomaterials to facilitate the delivery of "biologics" is a major area of research in cancer therapy. Here we designed and characterized a hyaluronic acid (HA) based self-assembling nanoparticles that can target CD44 receptors overexpressed on multidrug resistance (MDR) ovarian cancer. The nanoparticle system is composed of HA-poly(ethyleneimine)/HA-poly(ethylene glycol) (HA-PEI/HA-PEG) designed to deliver MDR1 siRNA for the treatment of MDR in an ovarian cancer model.HA-PEI/HA-PEG nanoparticles were synthesized and characterized, then the cellular uptake and knockdown efficiency of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles was further determined. A human xenograft MDR ovarian cancer model was established to evaluate the effects of the combination of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles and paclitaxel on MDR tumor growth.Our results demonstrated that HA-PEI/HA-PEG nanoparticles successfully targeted CD44 and delivered MDR1 siRNA into OVCAR8TR (established paclitaxel resistant) tumors. Additionally, HA-PEI/HA-PEG nanoparticles loaded with MDR1 siRNA efficiently down-regulated the expression of MDR1 and P-glycoprotein (Pgp), inhibited the functional activity of Pgp, and subsequently increased cell sensitivity to paclitaxel. HA-PEI/HA-PEG/MDR1 siRNA nanoparticle therapy followed by paclitaxel treatment inhibited tumor growth in MDR ovarian cancer mouse models.These findings suggest that this CD44 targeted HA-PEI/HA-PEG nanoparticle platform may be a clinicaly relevant gene delivery system for systemic siRNA-based anticancer therapeutics for the treatment of MDR cancers.
Project description:PurposeAstrocyte dysfunction is a hallmark of central nervous system injury or infection. As a primary contributor to neurodegeneration, astrocytes are an ideal therapeutic target to combat neurodegenerative conditions. Gene therapy has arisen as an innovative technique that provides excellent prospect for disease intervention. Poly (lactide-co-glycolide) (PLGA) and polyethylenimine (PEI) are polymeric nanoparticles commonly used in gene delivery, each manifesting their own set of advantages and disadvantages. As a clinically approved polymer by the Federal Drug Administration, well characterized for its biodegradability and biocompatibility, PLGA-based nanoparticles (PLGA-NPs) are appealing for translational gene delivery systems. However, our investigations revealed PLGA-NPs were ineffective at facilitating exogenous gene expression in primary human astrocytes, despite their success in other cell lines. Furthermore, PEI polymers illustrate high delivery efficiency but induce cytotoxicity. The purpose of this study is to develop viable and biocompatible NPsystem for astrocyte-targeted gene therapy.Materials and MethodsSuccessful gene expression by PLGA-NPs alone or in combination with arginine-modified PEI polymers (AnPn) was assessed by a luciferase reporter gene encapsulated in PLGA-NPs. Cytoplasmic release and nuclear localization of DNA were investigated using fluorescent confocal imaging with YOYO-labeled plasmid DNA (pDNA). NP-mediated cytotoxicity was assessed via lactate dehydrogenase in primary human astrocytes and neurons.ResultsConfocal imaging of YOYO-labeled pDNA confirmed PLGA-NPs delivered pDNA to the cytoplasm in a dose and time-dependent manner. However, co-staining revealed pDNA delivered by PLGA-NPs did not localize to the nucleus. The addition of AnPn significantly improved nuclear localization of pDNA and successfully achieved gene expression in primary human astrocytes. Moreover, these formulations were biocompatible with both astrocytes and neurons.ConclusionBy co-transfecting two polymeric NPs, we developed an improved system for gene delivery and expression in primary human astrocytes. These findings provide a basis for a biocompatible and clinically translatable method to regulate astrocyte function during neurodegenerative diseases and disorders.
Project description:Anticancer therapeutics employing RNA interference mechanism holds promising potentials for sequence-specific silencing of target genes. However targeted delivery of siRNAs to tumor tissues and cells and more importantly, their intracellular release at sites of interest still remains a major challenge that needs to be addressed before this technique could become a clinically viable option. In the current study, we have engineered and screened a series of CD44 targeting hyaluronic acid (HA) based self-assembling nanosystems for targeted siRNA delivery. The HA polymer was functionalized with lipids of varying carbon chain lengths/nitrogen content, as well as polyamines for assessing siRNA encapsulation. From the screens, several HA-derivatives were identified that could stably encapsulate/complex siRNAs and form self-assembled nanosystems, as determined by gel retardation assays and dynamic light scattering. Many HA derivatives could transfect siRNAs into cancer cells overexpressing CD44 receptors. Interestingly, blocking the CD44 receptors on the cells using free excess soluble HA prior to incubation of cy3-labeled-siRNA loaded HA nano-assemblies resulted in >90% inhibition of the receptor mediated uptake, confirming target specificity. In addition, SSB/PLK1 siRNA encapsulated in HA-PEI/PEG nanosystems demonstrated dose dependent and target specific gene knockdown in both sensitive and resistant A549 lung cancer cells overexpressing CD44 receptors. More importantly, these siRNA encapsulated nanosystems demonstrated tumor selective uptake and target specific gene knock down in vivo in solid tumors as well as in metastatic tumors. The HA based nanosystems thus portend to be promising siRNA delivery vectors for systemic targeting of CD44 overexpressing cancers including tumor initiating (stem-) cells and metastatic lesions.
Project description:Doxorubicin (DOX)-engineered poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) including phloretin (PHL) were designed and the feasible contribution of sialic acid (SA) to the improved tumor targeting and penetration capabilities was elucidated in lung adenocarcinoma models. DOX has been clinically used as liposomal formulations after its introduction to the inner side of vehicles, however DOX is anchored in the outer surface of PLGA NPs for improved tumor penetration by interactions with SA in this study. DOX (positively charged at physiological pH) was adsorbed onto the negatively charged PLGA NPs via electrostatic interactions and consequent binding of SA (negatively charged at physiological pH) to DOX located in NPs was also elucidated. DOX layer in DOX@PLGA NPs rendered improved endocytosis and partial contribution of SA (expressed in cancer cells) to that endocytosis was demonstrated. DOX@PLGA/PHL NPs provided enhanced antiproliferation potentials in A549 cells rather than single agent (DOX or PHL)-installed NPs. In addition, DOX-SA interactions seemed to play critical roles in tumor infiltration and accumulation of DOX@PLGA NPs in A549 tumor-xenografted mouse model. All these findings support the novel use of DOX which is used for the surface engineering of NPs for improved tumor targeting and penetration.