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1.
J Control Release ; 373: 319-335, 2024 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-38986911

RESUMO

Diabetic foot ulcer (DFU), which is characterised by damage to minute blood vessels or capillaries around wounds, is one of the most serious and dreaded complications of diabetes. It is challenging to repair chronic non-healing DFU wounds. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and promotes wound healing in DFU. However, it is difficult to sustainably deliver VEGF to the wound site owing to its poor stability and easy degradation. To overcome this challenge, lipid nanoparticles (LNP) encapsulating circular RNA (circRNA) encoding VEGF-A have been developed to continuously generate and release VEGF-A and accelerate diabetic wound healing. First, VEGF-A circRNA was synthesized using group I intron autocatalysis strategy and confirmed by enzyme digestion, polymerase chain reaction, and sequencing assay. VEGF-A circRNA was encapsulated in ionizable lipid U-105-derived LNP (U-LNP) using microfluidic technology to fabricate U-LNP/VEGF-A circRNA. For comparison, a commercially ionizable lipid ALC-0315-derived LNP (A-LNP) encapsulating circRNA (A-LNP/circRNA) was used. Dynamic light scattering and transmission electron microscopy characterization indicated that U-LNP/circRNA had spherical structure with an average diameter of 108.5 nm, a polydispersity index of 0.22, and a zeta potential of -3.31 mV. The messenger RNA (mRNA) encapsulation efficiency (EE%) of U-LNP was 87.12%. In vitro transfection data confirmed better stability and long-term VEGF-A expression of circRNA compared with linear mRNA. Assessment of cytotoxicity and innate immunity further revealed that U-LNP/circRNA was biocompatible and induced a weak congenital immune response. Cell scratch and angiogenesis tests demonstrated the bioactivity of U-LNP/VEGF-A circRNA owing to its VEGF-A expression. In situ bioluminescence imaging of firefly luciferase (F-Luc) probe and ELISA demonstrated that circRNA had long-term and strong expression of VEGF-A in the first week, and a gradual decrease in the next week at the wound site and surrounding areas. Finally, a diabetic mouse model was used to validate the healing effect of U-LNP/VEGF-A circRNA formulation. The results showed that a single dose of U-LNP/VEGF-A circRNA administered by dripping resulted in almost complete wound recovery on day 12, which was significantly superior to that of U-LNP/VEGF-A linear mRNA, and it also outperformed recombinant human vascular endothelial growth factor (rhVEGF) injection and A-LNP/circRNA dripping. Histological analysis confirmed the healing efficiency and low toxicity of U-LNP/VEGF-A circRNA formulation. Together, VEGF-A circRNA delivered by U-105-derived LNP showed good performance in wound healing, which was ascribed to the long-term expression and continuous release of VEGF-A, and has potential applications for the treatment of diabetic foot ulcer wounds.

2.
J Control Release ; 368: 663-675, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38492862

RESUMO

Interleukin-2 (IL-2) exhibits the unique capacity to modulate immune functions, potentially exerting antitumor effects by stimulating immune responses, making it highly promising for immunotherapy. However, the clinical use of recombinant IL-2 protein faces significant limitations due to its short half-life and systemic toxicity. To overcome these challenges and fully exploit IL-2's potential in tumor immunotherapy, this study reports the development of a tumor-activated IL-2 mRNA, delivered via lipid nanoparticles (LNPs). Initially, ionizable lipid U-101 derived nanoparticles (U-101-LNP) were prepared using microfluidic technology. Subsequent in vitro and in vivo delivery tests demonstrated that U-101-LNP achieved more effective transfection than the approved ALC-0315-LNP. Following this, IL-2F mRNAs, encoding fusion proteins comprising IL-2, a linker, and CD25 (IL-2Rα), were designed and synthesized through in vitro transcription. A cleavable linker, consisting of the peptide sequence SGRSEN↓IRTA, was selected for cleavage by matrix metalloproteinase-14 (MMP-14). IL-2F mRNA was then encapsulated in U-101-LNP to create U-101-LNP/IL-2F mRNA complexes. After optimization, assessments of expression efficiency, masking, and release characteristics revealed that IL-2F with linker C4 demonstrated superior performance. Finally, the antitumor activity of IL-2F mRNA was evaluated. The results indicated that U-101-LNP/IL-2F mRNA achieved the strongest antitumor effect, with an inhibition rate of 70.3%. Immunohistochemistry observations revealed significant expressions of IL-2, IFN-γ, and CD8, suggesting an up-regulation of immunomodulation in tumor tissues. This effect could be ascribed to the expression of IL-2F, followed by the cleavage of the linker under the action of MMP-14 in tumor tissue, which sustainably releases IL-2. H&E staining of tissues treated with U-101-LNP/IL-2F mRNA showed no abnormalities. Further evaluations indicated that the U-101-LNP/IL-2F mRNA group maintained proper levels of inflammatory factors without obvious alterations in liver and renal functions. Taken together, the U-101-LNP/IL-2F mRNA formulation demonstrated effective antitumor activity and safety, which suggests potential applicability in clinical immunotherapy.


Assuntos
Lipossomos , Nanopartículas , Neoplasias , Humanos , Interleucina-2/genética , Metaloproteinase 14 da Matriz , Imunoterapia , Neoplasias/terapia
3.
Int J Pharm ; 645: 123398, 2023 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-37690658

RESUMO

Polypeptides are a highly promising carrier for delivering hydrophobic drugs, due to their excellent biocompatibility, non-toxicity, and non-immunogenicity. Herein, a redox and pH dual-responsive poly(ethylene glycol)-SS-b-polypeptide micelles encapsulated with disulfide bridged paclitaxel-pentadecanoic acid prodrug was developed for cancer chemotherapy. First of all, disulfide bridged paclitaxel-pentadecanoic acid prodrug (PTX-SS-COOH) and poly(ethylene glycol)-SS-b-polylysine-b-polyphenylalanine (mPEG-SS-b-PLys-b-PPhe, ESLP) were synthesized and confirmed via NMR, MS, FT-IR or GPC. After that, PTX-SS-COOH (PSH) embedded mPEG-SS-b-PLys-b-PPhe (ESLP/PSH) micelles were prepared by mixing method based on electrostatic interactions and hydrophobic forces. For comparison, mPEG-b-PLys-b-PPhe (ELP) was mixed with PTX-SS-COOH to generate another kind of micelles (ELP/PSH). The characterization of ESLP/PSH micelles through dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed a spherical structure with a diameter of approximately 170 nm. It is noteworthy that ESLP/PSH micelles displayed a high drug-loading rate of 22.84%, and excellent stability, which can be attributed to the specific interactions between the prodrug and copolymer. Drug release analysis demonstrated that the micelles exhibited a substantial release of PTX in the presence of GSH at pH 5.0, indicating a pH and redox dual responsiveness. In vivo pharmacokinetic study revealed the ESLP/PSH micelles had increased bioavailability and an extended circulation time. Ultimately, antitumor efficacy and systemic toxicity evaluation in 4 T1 tumor-bearing mice confirmed that ESLP/PSH micelles achieved the highest level of tumor growth inhibition (ca. 83%) and the lowest systemic toxicity in comparison with ELP/PSH micelles and commercialized Taxol®. Taken together, the dual responsive micelles represent a promising PTX formulation with potential clinical application in cancer chemotherapy.


Assuntos
Neoplasias , Pró-Fármacos , Camundongos , Animais , Paclitaxel , Micelas , Espectroscopia de Infravermelho com Transformada de Fourier , Linhagem Celular Tumoral , Polietilenoglicóis/química , Peptídeos , Oxirredução , Dissulfetos , Concentração de Íons de Hidrogênio , Portadores de Fármacos/química
4.
Mol Pharm ; 20(10): 4971-4983, 2023 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-37699256

RESUMO

mRNA vaccines encoding a single spike protein effectively prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the emergence of SARS-CoV-2 variants leads to a wide range of immune evasion. Herein, a unique trivalent mRNA vaccine based on ancestral SARS-CoV-2, Delta, and Omicron variant spike receptor-binding domain (RBD) mRNAs was developed to tackle the immune evasion of the variants. First, three RBD mRNAs of SARS-CoV-2, Delta, and Omicron were coencapsulated into lipid nanoparticles (LNPs) by using microfluidic technology. After that, the physicochemical properties and time-dependent storage stability of the trivalent mRNA vaccine nanoformulation were tested by using dynamic light scattering (DLS). In vitro, the trivalent mRNA vaccine exhibited better lysosomal escape ability, transfection efficiency, and biocompatibility than did the commercial transfection reagent Lipo3000. In addition, Western blot analyses confirmed that the three RBD proteins can be detected in cells transfected with the trivalent mRNA vaccine. Furthermore, ex vivo imaging analysis indicated that the livers of BALB/c mice had the strongest protein expression levels after intramuscular (IM) injection. Using a prime-boost strategy, this trivalent vaccine elicited robust humoral and T-cell immune responses in both the high-dose and low-dose groups and showed no toxicity in BALB/c mice. Three specific IgG antibodies in the high-dose group against SARS-CoV-2, Delta, and Omicron variants approached ∼1/1,833,333, ∼1/1,866,667, and ∼1/925,000, respectively. Taken together, two doses of inoculation with the trivalent mRNA vaccine may provide broad and effective immunization responses against SARS-CoV-2 and variants.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Camundongos , Humanos , SARS-CoV-2/genética , Vacinas contra COVID-19 , COVID-19/prevenção & controle , Imunização , Vacinas de mRNA , Anticorpos Neutralizantes
5.
Int J Pharm ; 642: 123155, 2023 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-37402442

RESUMO

Lipid nanoparticles (LNPs)-based mRNA vaccines have shown great potential in the fight against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. However, it remains still a challenge to improve the delivery efficiency of LNPs and the long-term stability of their mediated mRNA vaccines. Herein, a novel ionizable lipid 2-hexyldecyl 6-(ethyl(3-((2-hexyldecyl)oxy)-2-hydroxypropyl)amino)hexanoate (HEAH) derived LNPs were developed for delivering the receptor binding domain (RBD) mRNAs. In vitro cell assays confirmed that the ionizable lipid HEAH with one ether bond and one ester bond derived LNPs possessed higher mRNA delivery efficiency compared with the approved ALC-0315 with two ester bonds used in the BNT162b2 vaccine. Notably, the HEAH-derived LNPs powder lyophilized did not significantly change for 30 days after storage at 37 °C indicating good thermostability. After two RBD mRNAs of Delta and Omicron variants were encapsulated into the HEAH-derived LNPs, a bivalent mRNA vaccine was obtained as a nanoparticle formulation. Importantly, the bivalent mRNA vaccine not only resisted Delta and Omicron and also generated protective antibodies against ancestral SARS-CoV-2. The HEAH-mediated bivalent vaccine induced stronger humoral and cellular immunity than those of the ALC-0315 group. Taken together, the ionizable lipid HEAH-derived LNPs show outstanding potential in improving the delivery efficiency of mRNA and the stability of mRNA vaccine.


Assuntos
COVID-19 , Nanopartículas , Humanos , Vacinas Combinadas , Vacina BNT162 , COVID-19/prevenção & controle , SARS-CoV-2/genética , Vacinas de mRNA , Decanoatos , Ésteres , Anticorpos Antivirais
6.
J Mater Chem B ; 11(31): 7454-7465, 2023 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-37448376

RESUMO

mRNA vaccines have emerged as a highly promising approach for preventing cancer and infectious diseases, attributed to their superior immunogenicity, rapid development speed, and quality-controlled scale production. While homologous mRNA vaccine administration is currently the most prevalent method employed in clinical settings, heterologous administration is a promising avenue worth exploring. In this report, two types of mRNA vaccine formulations for SARS-CoV-2 infection were developed based on different lipid nanoparticle (LNP) delivery systems, and heterologous and homologous mRNA vaccinations were administered to explore the levels of immune responses comparatively. First, five novel H-series ionizable lipids were synthesized and confirmed by NMR and MS. Subsequently, six SARS-CoV-2 receptor-binding domain (RBD) mRNA-encapsulated LNP formulations were prepared using a microfluidic mixer based on H-series and MC3 lipids. These formulations exhibited spherical structures with an average diameter ranging from 90-140 nm, as characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The safety of these formulations was confirmed in vitro by the cytotoxicity assay. Moreover, transfection assay, lysosomal escape test, and western blot, and in vivo biodistribution analyses collectively demonstrated that lipids H03 and MC3 exhibited superior in vitro and in vivo delivery efficacy in comparison to other H-series lipids. Notably, H03-Fluc mRNA exhibited an approximately 2.2-fold higher in vivo bioluminescence signal intensity than MC3-Fluc mRNA. Additionally, evaluation of humoral immunity demonstrated that homologous H03-mRNA vaccination elicited an immune response that was approximately 3-fold higher than that of homologous MC3-mRNA vaccination. More significantly, the heterologous H03-mRNA/MC3-mRNA vaccination elicited an immune response that was approximately 2-3-fold higher than that of homologous H03-mRNA vaccination and 6-9-fold higher than that of homologous MC3-mRNA vaccination, without any observable adverse effects. These results suggest that heterologous mRNA vaccination is superior to homologous mRNA vaccination and may be attributed to differences in LNP carriers. Therefore, our research may inspire further exploration of different delivery systems to enhance mRNA-based therapeutics.


Assuntos
COVID-19 , Humanos , COVID-19/prevenção & controle , SARS-CoV-2 , Distribuição Tecidual , Vacinas de mRNA , Lipídeos
7.
Med Sci Monit ; 29: e939972, 2023 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-37516906

RESUMO

Bone defects and dysfunctions are prevalent among patients, resulting from various causes such as trauma, tumors, congenital malformations, inflammation, and infection. The demand for bone defect repair materials is second only to blood transfusions. Artificial bone composites offer numerous advantages for bone damage repair, including their availability, absence of rejection or immune reactions, high malleability, exceptional mechanical strength, and outstanding biocompatibility. However, bacterial infections frequently occur during bone transplantation or on graft material structures, leading to severe complications such as osteomyelitis and osteoporosis. Moreover, existing osteogenic materials alone are inadequate to address the challenges posed by traumatic infections, presenting a significant hurdle for clinicians in reconstructing infectious bone defects. Consequently, it is crucial to functionalize artificial bone composites to facilitate effective bone repair and regeneration. Notably, antibacterial capabilities play a critical role in preventing and treating infectious bone defects, and current research is focusing on the interface between artificial bone composites and antibacterial treatments. This article provides an extensive review of the current state of artificial composite bone scaffolds with antibacterial properties for infection prevention in bone grafting.


Assuntos
Artrodese , Transplante Ósseo , Humanos , Osteogênese , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Inflamação
8.
Mol Pharm ; 20(5): 2513-2526, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37071030

RESUMO

Docetaxel (DTX), a semisynthetic analogue of paclitaxel, is often used to treat cancers. Owing to its poor aqueous solubility, the current formulation of DTX for clinical applications involves using high surfactant and ethanol concentrations, causing hypersensitivity reactions. To overcome this issue, we developed a reduction-responsive DTX prodrug encapsulated within human serum albumin (HSA) nanoparticles (DTX-SS-COOH/HSA NPs). First, the DTX prodrug was conjugated to undecanoic acid through a disulfide bond (DTX-SS-COOH) via a four-step reaction. Subsequently, DTX-SS-COOH/HSA NPs were prepared via the desolvation method. The NPs exhibited a spherical structure with a diameter range of 140-220 nm, as revealed by dynamic light scattering and transmission electron microscopy. Fluorescence quenching analysis confirmed the formation of DTX-SS-COOH/HSA, which was ascribed to electrostatic interactions and hydrophobic forces. Notably, NPs with a feed mole ratio corresponding to DTX-SS-COOH/HSA = 9:1 demonstrated high drug-loading and encapsulation efficiency of 12.84 and 93.11%, respectively, alongside good stability. Moreover, the reduced responsiveness experiment revealed an accelerated DTX release in the presence of glutathione. An in vivo pharmacokinetic study indicated that DTX-SS-COOH/HSA NPs demonstrated considerably a prolonged circulation time (6.2-fold) compared to that of free DTX. Ultimately, the antitumor test of MDA-MB-231 tumor-bearing mice revealed that DTX-SS-COOH/HSA NPs were superior to DTX/HSA NPs for tumor growth inhibition. Thus, DTX-SS-COOH/HSA NPs represent a promising DTX nanoformulation for clinical application.


Assuntos
Antineoplásicos , Nanopartículas , Neoplasias , Pró-Fármacos , Humanos , Camundongos , Animais , Docetaxel , Albumina Sérica Humana , Paclitaxel , Portadores de Fármacos/química , Nanopartículas/química , Linhagem Celular Tumoral , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Neoplasias/tratamento farmacológico
9.
Int J Pharm ; 636: 122822, 2023 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-36914018

RESUMO

Chemodynamic therapy (CDT) has emerged as a powerful tumor treatment option by inducing the imbalance of redox homeostasis in cancer cells. Nevertheless, the therapeutic outcomes were greatly limited because of insufficient endogenous H2O2 and upregulated cellular antioxidant defense in the tumor microenvironment (TME). Herein, a liposome-incorporated in-situ alginate hydrogel locoregional treatment strategy was developed, which involves using hemin-loaded artesunate dimer liposomes (HAD-LP) as redox-triggered self-amplified C-center free radical nanogenerator to enhance CDT. First, HAD-LP based on artesunate dimer glycerophosphocholine (ART-GPC) was prepared by a thin film method. Their spherical structure was manifested by dynamic light scattering (DLS) and transmission electron microscope (TEM). The generation of C-center free radicals from HAD-LP was carefully evaluated by using methylene blue (MB) degradation method. The results suggested that the hemin was reduced to heme under the action of glutathione (GSH), which could catalyze the breakage of endoperoxide of ART-GPC derived dihydroartemisinin (DHA) to generate toxic C-centered free radicals in a H2O2 and pH-independent manner. Moreover, the change of intracellular GSH and free radical level was monitored through ultraviolet spectroscopy and confocal laser scanning microscope (CLSM). It was revealed that the hemin reduction induced GSH depletion and elevated free radical level, disrupting cellular redox homeostasis. After co-incubation with MDA-MB-231 or 4 T1 cells, HAD-LP was found to be highly cytotoxic. In order to prolong retention and improve antitumor efficacy, HAD-LP was mixed with alginate and injected intratumorally into 4 T1 tumor bearing mice. The injected HAD-LP and alginate mixture formed in-situ hydrogel and achieved best antitumor effect with the growth inhibition rate of 72.6%. Together, the hemin-loaded artesunate dimer liposome-incorporated alginate hydrogel possessed effective antitumor activity through redox-triggered C-center free radical generation induced apoptosis in a H2O2 and pH-independent manner, which might be a promising candidate in the application of chemodynamic anti-tumor therapy.


Assuntos
Nanopartículas , Neoplasias , Animais , Camundongos , Artesunato , Lipossomos , Hidrogéis , Hemina , Peróxido de Hidrogênio , Alginatos , Glutationa , Concentração de Íons de Hidrogênio , Linhagem Celular Tumoral , Microambiente Tumoral
10.
J Mater Chem B ; 11(11): 2478-2489, 2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36843543

RESUMO

7-Ethyl-10-hydroxyl camptothecin (SN38), a semisynthetic derivative of camptothecin, exhibited extreme pharmacological activities in treating a range of cancers. However, its poor aqueous solubility and low stability hinder its clinical applications. Hence, a redox-responsive SN38 prodrug encapsulated human serum albumin (HSA) nanoparticle is developed to realize its potential in the clinic. First, a disulfide bond bridged 7-ethyl-10-hydroxyl camptothecin-undecanoic acid conjugate (SN38-SS-COOH) was synthesized and characterized structurally. After that, SN38-SS-COOH/HSA nanoparticles (SNH NPs) were prepared by the desolvation method. The SNH NPs with a feed molar ratio of 9 : 1 of SN38-SS-COOH : HSA showed a spherical structure with a diameter range of approximately 120-150 nm revealed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Fluorescence quenching confirmed the formation of SNH NP complexes by dual hydrophobic force and electrostatic interaction. The SNH NPs have a high drug loading of 10.44% and an encapsulation efficiency of 89.59% with good stability. Moreover, the redox responsiveness was validated by glutathione (GSH)-triggered accelerated release of parent drug SN38. In an in vivo pharmacokinetic study, the SNH NPs exhibited a significantly prolonged circulation time (t1/2, 3.77-fold) compared with free SN38. Finally, the in vivo antitumor efficacy and systemic toxicity of SNH NPs in a breast xenograft model were thoroughly evaluated. The inhibition rate of tumor growth induced by the SNH NPs reached 70.1%, while only 50.1% was achieved for irinotecan at an equivalent SN38 dosage of 10 mg kg-1. More importantly, the SNH NPs achieved a higher level of tumor growth inhibition (85.3%) by increasing the dosage to 60 mg kg-1 SN38 without obvious adverse effects. Taken together, the use of redox-responsive SN38 prodrug/HSA NPs could be a promising strategy to deliver highly active SN38 for breast cancer chemotherapy.


Assuntos
Antineoplásicos Fitogênicos , Neoplasias da Mama , Nanopartículas , Pró-Fármacos , Humanos , Feminino , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Albumina Sérica Humana , Pró-Fármacos/química , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Nanopartículas/química , Dissulfetos
11.
Int J Pharm ; 635: 122761, 2023 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-36822341

RESUMO

Human serum albumin (HSA) is an important nanocarrier of hydrophobic drugs due to its biocompatibility, bioresorbability, non-immunogenicity and intrinsic targetability. However, HSA/drug nanocomplexes have to experience complicated manufacturing process including multiple high-pressure homogenization and removing organic solvent under reduced pressure condition. Besides, the clinical application of these HSA/drug nanocomplexes is often limited because of their unsatisfactory stability and restricted dose. To overcome these issues, a redox-responsive paclitaxel-pentadecanoic acid prodrug conjugate embedded human serum albumin nanoparticles (NPs) was developed as a model in this report. First, PTX was activated and conjugated with 11-mercaptoundecanoic acid through a disulfide bond. The resultant disulfide bond bridged paclitaxel-pentadecanoic acid conjugate (PTX-SS-C10-COOH) was characterized by NMR and MS. After that, PTX-SS-C10-COOH dissolved in ethanol was mixed with HSA in water followed by lyophilization to generate HSA/PTX-SS-C10-COOH nanoparticles (HPTX NPs). Dynamic light scattering (DLS) and transmission electron microscopy (TEM) characterization indicated that the HPTX NPs have spherical structure with an average diameter of approximately 120 nm. The formation of HSA/PTX-SS-C10-COOH NPs was confirmed by fluorescence quenching technology, ascribed to electrostatic and hydrophobic interactions. The HPTX NPs displayed a highdrug loading of 29.78 % and an entrapment efficiency of 94.16 %. Their reduced responsiveness was validated by glutathione (GSH)-triggered fast release of PTX. The pharmacokinetics, antitumor efficacy and systemic toxicity of HPTX NPs were thoroughly evaluated. The results showed that the HPTX NPs had longer retention, more effective tumor growth inhibition and lower toxicity compared with commercialized Taxol®. Importantly, the HPTX NPs could be administered at much high dose to achieve a significant tumor growth inhibition compared with Abraxane®. Together, the redox-responsive HPTX NPs with high drug loading is a promising strategy to deliver PTX for cancer chemotherapy.


Assuntos
Nanopartículas , Neoplasias , Humanos , Paclitaxel , Albumina Sérica Humana , Nanopartículas/química , Neoplasias/tratamento farmacológico , Oxirredução , Dissulfetos , Linhagem Celular Tumoral
12.
Int J Pharm ; 632: 122565, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36586634

RESUMO

Diabetes is often accompanied by chronic non-healing wounds, and vascularendothelial growth factor A (VEGF-A) is crucial in the treatment of chronic diabetic wounds. However, the application of VEGF-A protein in clinic is limited due to poor absorption and short half-life of protein macromolecule. Herein, we employed an emerging protein replacement therapy by delivering VEGF-A mRNA into the body to express the desired protein to accelerate diabetic wound healing. Primarily, VEGF-A mRNA was synthesized by an in vitro transcription (IVT) method and encapsulated with an ionizable lipid-mediated nanoparticles (LNP) delivery system via a microfluidic method. The resultant LNP/VEGF-A mRNA were characterized by using dynamic light scattering (DLS) and transmission electron microscope(TEM). The nanoparticles have regular spherical morphology with an average particle size of 101.17 nm, a narrow polydispersity (PDI) of 0.17 and negative Zeta potential of -3.05 mV. The bioactivities of the nanoparticles formulation were evaluated against HUVEC cells through cell proliferation, migration and tube formation assays. It was found that the LNP/VEGF-A mRNA nanoparticles could promote endothelial cell proliferation. In addition, they exhibited successful mRNA delivery and high VEGF-A protein expression in vitro and in vivo by means of Western Blot assay and in vivo imaging system (IVIS). Finally, C57BL/6 diabetic mice model was established and intradermally treated with the LNP/VEGF-A mRNA nanoparticles. It was found that the LNP/VEGF-A mRNA treated wounds were almost healed after 14 days with an average wound area of 2.4 %, compared with the PBS group of 21.4 %. Apparently, the nanoparticles formulation was able to significantly expedite diabetic wound healing. The histological analysis containing H&E, Masson's trichrome staining and CD31 further confirmed the healing efficacy and low toxicity of the formulation. Taken together, the LNP/VEGF-A mRNA nanoparticles can be taken up by cells to express protein effectively and improve diabetic wound healing, which might have potential application in the treatment of chronic diabetic wounds as a protein replacement therapy.


Assuntos
Diabetes Mellitus Experimental , Nanopartículas , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Diabetes Mellitus Experimental/terapia , Camundongos Endogâmicos C57BL , Cicatrização
13.
Bioorg Med Chem ; 78: 117135, 2023 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-36577327

RESUMO

Vascular endothelial growth factor A (VEGFA) plays an important role in the healing of skin wound. However, the application of VEGFA protein in clinic is limited because of its high cost manufacturing, complicated purification and poor pharmacokinetic profile. Herein, we developed nucleoside-modified mRNA encoding VEGFA encapsulated ionizable lipid nanoparticles (LNP) to improve angiogenesis and increase wound healing rate. First, VEGFA mRNA was synthesized by an in vitro transcription (IVT) method. After that, VEGFA mRNA-LNP was prepared by encapsulating mRNA in ionizable lipid based nanoparticles via a microfluidic mixer. The physicochemical properties of VEGFA mRNA-LNP were investigated via dynamic light scattering (DLS) and transmission electron microscopy (TEM). The results showed that the VEGFA mRNA-LNP possessed regular spherical morphology with an average size of 112.67 nm and a negative Zeta potential of -3.43 mV. The LNP delivery system had excellent lysosome escape capability and high transfection efficiency. ELISA and Western Blot analysis indicated that the mRNA-LNP could express VEGFA protein in Human umbilical vein endothelial cells (HUVECs). Besides, endothelial tube formation, cell proliferation and scratch assays were performed. The results revealed VEGFA mRNA-LNP boosted angiogenesis, cell proliferation and cell migration by expressing VEGFA protein. Finally, C57BL/6 mouse model of skin wound was established and intradermally treated with VEGFA mRNA-LNP. The VEGFA mRNA-LNP treated wounds were almost healed with an average wound size of 1.56 mm2 compared with the blank of 18.66 mm2 after 9 days. The results indicated that the VEGFA mRNA-LNP was able to significantly expedite wound healing. Histological analysis further demonstrated tissue epithelialization, collagen deposition and enhancement of vascular density after treatment. Taken together, VEGFA mRNA-LNP can be uptaken by cells to express protein effectively and promote wound healing, which may provide a promising strategy for clinical remedy.


Assuntos
Nanopartículas , Fator A de Crescimento do Endotélio Vascular , Camundongos , Animais , Humanos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , RNA Mensageiro/genética , Camundongos Endogâmicos C57BL , Cicatrização , Nanopartículas/química , Células Endoteliais da Veia Umbilical Humana/metabolismo
14.
Nanomaterials (Basel) ; 12(20)2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36296881

RESUMO

In this research, we constructed a novel engineered tripeptide modified with lipoic acid (LA-RWR), followed by crosslinking of lipoic acid to form nanoparticles (c-LA-RWR). LA-RWR was also modified with phenethylamine (PEA) on the C-terminus to achieve better antibacterial activities. The as-prepared c-LA-RWR and LA-RWR-PEA were effective against E.coli, S.aureus, C.albicans, and methicillin-resistant Staphylococcus aureus, with minimum inhibitory concentration values ranging from 2 to 16 µg/mL, which greatly improved the performance of LA-RWR. Similar antibacterial activities were demonstrated in anti-biofilm activity; there was no matter on the biofilm that was already established or forming. Moreover, c-LA-RWR/LA-RWR-PEA remarkably induced cytoplasmic membrane depolarization and outer membrane permeabilization, resulting in varying degrees of damage to the bacterial morphology, which were consistent with the results obtained via electron microscopy. Thus, our results show that c-LA-RWR/LA-RWR-PEA exhibited excellent efficacy against a variety of microorganisms with good biosafety, providing new strategies by which to improve the performance of antimicrobial peptides.

15.
Mol Pharm ; 19(12): 4588-4600, 2022 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-35731922

RESUMO

Protein kinase N3 (PKN3), by virtue of its abnormal expression in prostate cells, has been widely used as a target of RNAi (shRNA, siRNA, miRNA) therapy. The major challenges of PKN3 RNAi therapy lie in how to design effective interference sequences and delivery systems. Herein, new PKN3 shRNA sequences (shPKN3-2459 and shPKN3-3357) were designed, and bioreducible, biodegradable, ionizable lipid-based nanoparticles were developed for shPKN3 delivery. First, an ionizable lipid (DDA-SS-DMA) bridged with disulfide bond and ester bonds was synthesized by a three-step reaction and confirmed by MS, 1H NMR, and 13C NMR. The ionizable lipid was mixed with cholesterol, DSPC, PEG-lipid, and shPKN3 by a microfluidic mixer to prepare lipid nanoparticles (LNP-shPKN3) which were characterized by DLS and TEM. Afterward, the pH and glutathione (GSH)-responsiveness of the DDA-SS-DMA based LNP delivery system were investigated by lysosome escape and gel electrophoresis assays. Compared with the commercial transfection reagent Lipo2000, the DDA-SS-DMA based delivery system showed higher transfection efficiency and lower toxicity. Western blot analysis, invasion tests, and migration assays were performed to evaluate the silencing effect of shPKN3 in vitro. In in vivo studies, high tumor suppression (65.8%) and treatment safety were evident in the LNP-shPKN3-2459 treatment group. Taken together, the DDA-SS-DMA based delivery system encapsulating shPKN3-2459 showed significant antitumor efficacy and might be a promising formulation for the treatment of prostate cancer.


Assuntos
Nanopartículas , Neoplasias da Próstata , Humanos , Masculino , Lipídeos/química , Nanopartículas/química , Próstata , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , RNA Interferente Pequeno
16.
Bioorg Med Chem ; 69: 116884, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35752145

RESUMO

Protein kinase N3 (PKN3), an AGC-family member, is often overexpressed in breast tumor cells. RNAi therapy is a promising approach to inhibit tumor growth by reducing the expression of PKN3. In this report, lipid nanoparticles encapsulated with new shRNA PKN3 (SS-LNP/shPKN3) with redox-responsiveness were developed in order to specifically down-regulate the expression of PKN3 for breast cancer treatment. The SS-LNP/shPKN3 was prepared by microfluidic method using disulfide bonds based ionizable lipid as main component. The as-prepared SS-LNP/shPKN3 lipid nanoparticles were characterized via using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The results indicated that the obtained SS-LNP/shPKN3 exhibited uniform particle size and regular spherical morphology. Moreover, glutathione (GSH) triggered release of shPKN3 confirmed the redox-responsiveness of the SS-LNP/shPKN3. Finally, the anti-tumor effect of SS-LNP/shPKN3 was evaluated against MDA-MB-231 cells and derived xenograft tumor bearing mice. It was found that the SS-LNP/shPKN3-2 had the highest PKN3 protein inhibition rate of 60.8% and tumor inhibition rate of 62.3%. Taken together, the SS-LNP/shPKN3 might be a potential therapeutic strategy for breast cancer.


Assuntos
Neoplasias da Mama , Nanopartículas , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Feminino , Humanos , Lipossomos , Camundongos , Nanopartículas/química , Proteína Quinase C , RNA Interferente Pequeno/química
17.
Int J Pharm ; 619: 121714, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35367585

RESUMO

Dimeric artesunate phospholipid (ART-GPC), an amphiphilic derivative of artemisinin dimer reported in our previous work, can be applied to treat malaria effectively. The objective of this study is to develop a facile method for the industrial production of ART-GPC liposomes. Conventional methods including thin film hydration (TFH), ethanol injection (EI), and freeze drying (FD) were used to prepare ART-GPC liposomes, and the resultants presented poor physicochemical properties. Fortunately, a modified thin film hydration method (MTFH) by forming thin film of ART-GPC composed of fine lipid bilayer structure in the vials showed promise for the liposomes production. A quality design strategy (solvents, pressure, hydration time, and temperature) was performed to obtain optimal physicochemical characteristics and production conditions. Thereafter, ART-GPC liposomes are produced under GMP conditions with the size of 176.32 nm, PDI of 0.17, zeta potential of -25.79 mV, and osmotic pressure of 297.33 mOsm/kg, confirming the scalability and reproductivity of the MTFH technology. It is the first report that the MTFH method allows liposomes to be preserved in a dry film state and in-situ hydrated in injection vials with excellent performance. Conclusively, the MTFH method is a promising technology for the large-scale production of ART-GPC liposomes.


Assuntos
Antimaláricos , Lipossomos , Artesunato/química , Fenômenos Químicos , Lipossomos/química , Fosfolipídeos/química , Polímeros
18.
Antimicrob Agents Chemother ; 66(5): e0206521, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35416709

RESUMO

Current best practice for the treatment of malaria relies on short half-life artemisinins that are failing against emerging Kelch 13 mutant parasite strains. Here, we introduce a liposome-like self-assembly of a dimeric artesunate glycerophosphocholine conjugate (dAPC-S) as an amphiphilic prodrug for the short-lived antimalarial drug, dihydroartemisinin (DHA), with enhanced killing of Kelch 13 mutant artemisinin-resistant parasites. Cryo-electron microscopy (cryoEM) images and the dynamic light scattering (DLS) technique show that dAPC-S typically exhibits a multilamellar liposomal structure with a size distribution similar to that of the liposomes generated using thin-film dispersion (dAPC-L). Liquid chromatography-mass spectrometry (LCMS) was used to monitor the release of DHA. Sustainable release of DHA from dAPC-S and dAPC-L assemblies increased the effective dose and thus efficacy against Kelch 13 mutant artemisinin-resistant parasites in an in vitro assay. To better understand the enhanced killing effect, we investigated processes for deactivation of both the assemblies and DHA, including the roles of serum components and trace levels of iron. Analysis of parasite proteostasis pathways revealed that dAPC assemblies exert their activity via the same mechanism as DHA. We conclude that this easily prepared multilamellar liposome-like dAPC-S with long-acting efficacy shows potential for the treatment of severe and artemisinin-resistant malaria.


Assuntos
Antimaláricos , Artemisininas , Malária Falciparum , Malária , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artemisininas/farmacologia , Artemisininas/uso terapêutico , Artesunato/farmacologia , Artesunato/uso terapêutico , Microscopia Crioeletrônica , Resistência a Medicamentos/genética , Humanos , Lipossomos/química , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Plasmodium falciparum/genética
19.
Mol Pharm ; 18(10): 3862-3870, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34470216

RESUMO

In this work, dimeric artesunate-phosphatidylcholine conjugate (dARTPC)-based liposomes encapsulated with irinotecan (Ir) were developed for anticancer combination therapy. First, dARTPC featured with unique amphipathic properties formed liposomes by classical thin-film methods. After that, Ir was encapsulated into dARTPC-based liposomes (Ir/dARTPC-LP) by the triethylammonium sucrose octasulfate gradient method. Physicochemical characterization indicated that Ir/dARTPC-LP had a mean size of around 140 nm and a negative ζ potential of approximately -30 mV. Most noticeably, liposomes displayed an encapsulation efficiency of greater than 98% with a controllable drug loading of 4-22%. The in vitro release of dihydroartemisinin (DHA) and Ir from Ir/dARTPC-LP was investigated by dialysis in different media. It was found that effective release of both DHA (65.42%) and Ir (77.28%) in a weakly acidic medium (pH 5.0) after 48 h was achieved in comparison to very slow release under a neutral environment (DHA 9.90% and Ir 8.72%), indicating the controllable release of both drugs. Confocal laser scanning microscopy confirmed the improved cellular internalization of Ir/dARTPC-LP. The cytotoxicity of Ir/dARTPC-LP was evaluated in the MCF-7, A549, and HepG2 cell lines. The results showed that Ir/dARTPC-LP had significant synergistic efficacy in the loss of cell growth. In vivo anticancer evaluation was performed using a 4T1 xenograft tumor model. Ir/dARTPC-LP had a high tumor inhibition rate of 62.7% without significant toxicity in comparison with the injection of Ir solution. Taken together, dARTPC encapsulated with Ir has great potential for anticancer combination therapy.


Assuntos
Artesunato/administração & dosagem , Sistemas de Liberação de Medicamentos , Irinotecano/administração & dosagem , Lipossomos/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Artesunato/farmacocinética , Artesunato/uso terapêutico , Linhagem Celular Tumoral , Combinação de Medicamentos , Sistemas de Liberação de Medicamentos/métodos , Feminino , Irinotecano/farmacocinética , Irinotecano/uso terapêutico , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/tratamento farmacológico , Fosfatidilcolinas
20.
Int J Pharm ; 609: 121138, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34592395

RESUMO

Artesunate (ARS) is the only artemisinin-based intravenous drug approved for treatment of malaria in the clinic. ARS is rapidly metabolized in vivo to short lived (∼30-45 min) but fast acting, dihydroartemisinin (DHA). The short half-life of DHA necessitates multiple dose administration to circumvent the risk of recrudescence and development of artemisinin resistance. In this work, we report a stable, safe and potent alternative artemisinin-based injectable nanocomplex consisting of dimeric artesunate-choline conjugate (dACC) micelles coated with hyaluronic acid (HA). Firstly, dACC was synthesized by one-step esterification of two artesunate molecules with 3-(dimethylamino)-1,2-propanediol followed by quaternization. After that, dACC was self-assembled into cationic nanomicelles and further coated with anionic small molecular weight HA. The HA-coated dACC nanocomplex (dACC/HA nanocomplex) has a narrow size distribution of about 30 nm. Hemolytic toxicity and cytotoxicity studies revealed a favorable bio-safety profile. Finally, in vitro and in vivo studies showed the dACC/HA nanocomplex possess superior safety and antimalarial efficacy compared to ARS. Taken together, the dACC/HA nanocomplex is a promising injectable alternative to the traditional clinically used artesunate.


Assuntos
Antimaláricos , Malária Falciparum , Antimaláricos/uso terapêutico , Artesunato , Colina , Humanos , Ácido Hialurônico/uso terapêutico , Malária Falciparum/tratamento farmacológico , Micelas
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