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1.
Nano Lett ; 22(4): 1511-1517, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35148107

RESUMO

Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-associated virus (AAV) vectors, which are the most widely used viral vectors for in vivo gene therapy, are typically characterized using PCR, ELISA, and analytical ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as charge-detection mass spectroscopy, static light scattering, and mass photometry offer turnaround times of minutes for measuring AAV mass using optical or charge properties of AAV. Here, we demonstrate an orthogonal method where suspended nanomechanical resonators (SNR) are used to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a precision near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.


Assuntos
Dependovirus , Vetores Genéticos , Capsídeo , DNA , Dependovirus/genética , Vetores Genéticos/genética
2.
Proc Natl Acad Sci U S A ; 116(33): 16473-16478, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31358641

RESUMO

Sustained exposure of lymphoid tissues to vaccine antigens promotes humoral immunity, but traditional bolus immunizations lead to rapid antigen clearance. We describe a technology to tailor vaccine kinetics in a needle-free platform translatable to human immunization. Solid pyramidal microneedle (MN) arrays were fabricated with silk fibroin protein tips encapsulating a stabilized HIV envelope trimer immunogen and adjuvant, supported on a dissolving polymer base. Upon brief skin application, vaccine-loaded silk tips are implanted in the epidermis/upper dermis where they release vaccine over a time period determined by the crystallinity of the silk matrix. Following MN immunization in mice, Env trimer was released over 2 wk in the skin, correlating with increased germinal center (GC) B cell responses, a ∼1,300-fold increase in serum IgG titers and a 16-fold increase in bone marrow (BM) plasma cells compared with bolus immunization. Thus, implantable MNs provide a practical means to substantially enhance humoral immunity to subunit vaccines.


Assuntos
Preparações de Ação Retardada/farmacologia , Imunidade Humoral , Agulhas , Próteses e Implantes , Vacinação , Animais , Formação de Anticorpos/imunologia , Antígenos/imunologia , Bombyx , Centro Germinativo/imunologia , Linfonodos/imunologia , Camundongos Endogâmicos BALB C , Seda , Pele
3.
Proc Natl Acad Sci U S A ; 115(12): E2696-E2705, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29432194

RESUMO

Small interfering RNA (siRNA) represents a promising class of inhibitors in both fundamental research and the clinic. Numerous delivery vehicles have been developed to facilitate siRNA delivery. Nevertheless, achieving highly potent RNA interference (RNAi) toward clinical translation requires efficient formation of RNA-induced gene-silencing complex (RISC) in the cytoplasm. Here we coencapsulate siRNA and the central RNAi effector protein Argonaute 2 (Ago2) via different delivery carriers as a platform to augment RNAi. The physical clustering between siRNA and Ago2 is found to be indispensable for enhanced RNAi. Moreover, by utilizing polyamines bearing the same backbone but distinct cationic side-group arrangements of ethylene diamine repeats as the delivery vehicles, we find that the molecular structure of these polyamines modulates the degree of siRNA/Ago2-mediated improvement of RNAi. We apply this strategy to silence the oncogene STAT3 and significantly prolong survival in mice challenged with melanoma. Our findings suggest a paradigm for RNAi via the synergistic coassembly of RNA with helper proteins.


Assuntos
Proteínas Argonautas/genética , Terapia Genética/métodos , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Complexo de Inativação Induzido por RNA/química , Animais , Proteínas Argonautas/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Melanoma Experimental/genética , Melanoma Experimental/mortalidade , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Oncogenes/genética , Poliaminas/química , RNA Antissenso/administração & dosagem , RNA Antissenso/farmacologia , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Mensageiro , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Fator de Transcrição STAT3/genética , Relação Estrutura-Atividade , Transfecção/métodos
4.
Biomacromolecules ; 21(2): 566-580, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31846304

RESUMO

Expanding the toolkit of modular and functional synthetic material systems for biomimetic extracellular matrices (ECMs) is needed for achieving more predictable and characterizable cell culture. In the present study, we engineered a synthetic hydrogel system incorporating poly(γ-propargyl-l-glutamate) (PPLG), an N-carboxy anhydride polypeptide with a unique α-helical secondary structure. PPLG macromers were cross-linked into poly(ethylene glycol) (PEG) networks to form hybrid polypeptide-PEG hydrogels. We compared the properties of PPLG-PEG to systems where the PPLG macromers were replaced with 8-arm PEG or poly(γ-propargyl-d,l-glutamate) (PPDLG), which has a flexible random-coil conformation. We evaluated each hydrogel system as synthetic ECMs for two-dimensional (2D) endothelial cell culture. Cells on PPLG-PEG displayed superior attachment and spreading at comparable adhesion ligand incorporation concentrations, demonstrating the unique benefit of combining the more rigid and hydrophobic α-helical PPLG within the more flexible and hydrophilic PEG matrix. The modular PPLG macromer is a promising building block for developing other types of PPLG-based hydrogels with favorable and tunable properties.


Assuntos
Técnicas de Cultura de Células/instrumentação , Matriz Extracelular/química , Hidrogéis/química , Peptídeos/química , Adesão Celular , Técnicas de Cultura de Células/métodos , Células Cultivadas , Recuperação de Fluorescência Após Fotodegradação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células-Tronco Pluripotentes Induzidas/citologia , Permeabilidade
5.
Angew Chem Int Ed Engl ; 59(7): 2776-2783, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31747099

RESUMO

Layer-by-layer nanoparticles (NPs) are modular drug delivery vehicles that incorporate multiple functional materials through sequential deposition of polyelectrolytes onto charged nanoparticle cores. Herein, we combined the multicomponent features and tumor targeting capabilities of layer-by-layer assembly with functional biosensing peptides to create a new class of nanotheranostics. These NPs encapsulate a high weight percentage of siRNA while also carrying a synthetic biosensing peptide on the surface that is cleaved into a urinary reporter upon exposure to specific proteases overexpressed in the tumor microenvironment. Importantly, this biosensor reports back on a molecular signature characteristic to metastatic tumors and associated with poor prognosis, MMP9 protease overexpression. This nanotheranostic mediates noninvasive urinary-based diagnostics in mouse models of three different cancers with simultaneous gene silencing in flank and metastatic mouse models of ovarian cancer.


Assuntos
Neoplasias Colorretais/diagnóstico , Nanopartículas/química , Neoplasias Ovarianas/diagnóstico , Peptídeos/química , Nanomedicina Teranóstica , Animais , Técnicas Biossensoriais , Neoplasias Colorretais/genética , Sistemas de Liberação de Medicamentos , Feminino , Inativação Gênica , Camundongos , Neoplasias Ovarianas/genética , Peptídeos/síntese química
6.
J Am Chem Soc ; 141(37): 14736-14741, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31460760

RESUMO

Preparation of an unzipping polyester is reported. The monomer was prepared from benzoic acid in a four-step sequence. Step growth polymerization of the monomer provides the target polymer. Efficient depolymerization upon irradiation at 254 nm was confirmed with a quantum yield of >0.8. The photolysis mechanism was investigated, and the results of radical trapping experiments are consistent with an initial Norrish type I like homolysis followed by a radical mediated depropagation reaction driven by aromatization.

7.
Angew Chem Int Ed Engl ; 56(44): 13709-13712, 2017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-28925033

RESUMO

Messenger RNA (mRNA) represents a promising class of nucleic acid drugs. Although numerous carriers have been developed for mRNA delivery, the inefficient mRNA expression inside cells remains a major challenge. Inspired by the dependence of mRNA on 3'-terminal polyadenosine nucleotides (poly A) and poly A binding proteins (PABPs) for optimal expression, we complexed synthetic mRNA containing a poly A tail with PABPs in a stoichiometric manner and stabilized the ribonucleoproteins (RNPs) with a family of polypeptides bearing different arrangements of cationic side groups. We found that the molecular structure of these polypeptides modulates the degree of PABP-mediated enhancement of mRNA expression. This strategy elicits an up to 20-fold increase in mRNA expression in vitro and an approximately fourfold increase in mice. These findings suggest a set of new design principles for gene delivery by the synergistic co-assembly of mRNA with helper proteins.


Assuntos
Peptídeos/química , Poli A/química , Proteínas de Ligação a Poli(A)/química , Poliaminas/química , RNA Mensageiro/administração & dosagem , Transfecção/métodos , Animais , Expressão Gênica , Células HEK293 , Humanos , Camundongos , RNA Mensageiro/química , RNA Mensageiro/genética , Transgenes
8.
J Physiol ; 590(15): 3495-505, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22641777

RESUMO

Recent studies have suggested the presence of cardiac atrophy as a key component of the pathogenesis of the postural orthostatic tachycardia syndrome (POTS), similar to physical deconditioning. It has also been shown that exercise intolerance is associated with a reduced stroke volume (SV) in POTS, and that the high heart rate (HR) observed at rest and during exercise in these patients is due to this low SV. We tested the hypotheses that (a) circulatory control during exercise is normal in POTS; and (b) that physical 'reconditioning' with exercise training improves exercise performance in patients with POTS. Nineteen (18 women) POTS patients completed a 3 month training programme. Cardiovascular responses during maximal exercise testing were assessed in the upright position before and after training. Resting left ventricular diastolic function was evaluated by Doppler echocardiography. Results were compared with those of 10 well-matched healthy sedentary controls. A lower SV resulted in a higher HR in POTS at any given oxygen uptake (V(O(2))) during exercise while the cardiac output (Q(c))-V(O(2)) relationship was normal. V(O(2peak)) was lower in POTS than controls (26.1 ± 1.0 (SEM) vs. 36.3 ± 0.9 ml kg-1 min-1; P < 0.001) due to a lower peak SV (65 ± 3 vs. 80 ± 5 ml; P = 0.009). After training in POTS, HR became lower at any given due to increased SV without changes in the ­ relationship. V(O(2peak)) increased by 11% (P < 0.001) due to increased peak SV (P = 0.021) and was proportional to total blood volume. Peak HR was similar, but HR recovery from exercise was faster after training than before training (P = 0.036 for training and 0.009 for interaction). Resting diastolic function was mostly normal in POTS before training, though diastolic suction was impaired (P = 0.023). There were no changes in any Doppler index after training. These results suggest that short-term exercise training improves physical fitness and cardiovascular responses during exercise in patients with POTS.


Assuntos
Exercício Físico/fisiologia , Síndrome da Taquicardia Postural Ortostática/fisiopatologia , Adulto , Terapia por Exercício , Feminino , Frequência Cardíaca/fisiologia , Humanos , Masculino , Volume Sistólico , Função Ventricular Esquerda/fisiologia
9.
ACS Nano ; 12(7): 6504-6514, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29944833

RESUMO

The delivery of small interfering RNA (siRNA) remains a major hurdle for the clinical translation of RNA interference (RNAi) therapeutics. Because of its low valency and rigid nature, siRNA typically requires high excesses of cationic delivery materials to package it stably and deliver it to the cytoplasm of target cells, resulting in high toxicities and inefficient gene silencing in vivo. To address these challenges, we pair a polymeric form of siRNA, p-shRNA, with optimized biodegradable polycations to form stable complexes that induce far more potent gene silencing than with siRNA complexes. Furthermore, we unveil a set of design rules governing p-shRNA delivery, using degradable polycations containing hydrophobic and stabilizing polyethylene glycol domains that enable both stable condensation and efficient release inside cells. We demonstrate the therapeutic potential of this approach by silencing the oncogene STAT3 in a well-established B16F10 mouse melanoma model to significantly prolong survival. By blending nucleic acid engineering and polymer design, our system provides a potentially translatable platform for RNAi-based therapies.


Assuntos
Técnicas de Transferência de Genes , Poliaminas/química , Polímeros/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Aminação , Animais , Linhagem Celular Tumoral , Esterificação , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Polieletrólitos , Polietilenoglicóis/química , RNA Interferente Pequeno/genética
10.
ACS Nano ; 11(3): 2531-2544, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28157292

RESUMO

Messenger RNA (mRNA) represents a promising class of nucleic-acid-based therapeutics. While numerous nanocarriers have been developed for mRNA delivery, the inherent labile nature of mRNA results in a very low transfection efficiency and poor expression of desired protein. Here we preassemble the mRNA translation initiation structure through an inherent molecular recognition between 7-methylguanosine (m7G)-capped mRNA and eukaryotic initiation factor 4E (eIF4E) protein to form ribonucleoproteins (RNPs), thereby mimicking the first step of protein synthesis inside cells. Subsequent electrostatic stabilization of RNPs with structurally tunable cationic carriers leads to nanosized complexes (nanoplexes), which elicit high levels of mRNA transfection in different cell types by enhancing intracellular mRNA stability and protein synthesis. By investigating a family of synthetic polypeptides bearing different side group arrangements of cationic charge, we find that the molecular structure modulates the nanoscale distance between the mRNA strand and the eIF4E protein inside the nanoplex, which directly impacts the enhancement of mRNA transfection. To demonstrate the biomedical potential of this approach, we use this approach to introduce mRNA/eIF4E nanoplexes to murine dendritic cells, resulting in increased activation of cytotoxic CD8 T cells ex vivo. More importantly, eIF4E enhances gene expression in lungs following a systemic delivery of luciferase mRNA/eIF4E in mice. Collectively, this bioinspired molecular assembly method could lead to a new paradigm of gene delivery.


Assuntos
Fator de Iniciação 4E em Eucariotos/genética , Técnicas de Transferência de Genes , Nanopartículas/química , Iniciação Traducional da Cadeia Peptídica/genética , Capuzes de RNA/genética , RNA Mensageiro/genética , Fator de Iniciação 4E em Eucariotos/química , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/química
11.
ACS Biomater Sci Eng ; 1(3): 148-156, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33429517

RESUMO

Uncontrolled bleeding and infection are the major causes of death and morbidity from traumatic wounds during military conflicts, disasters, and accidents. Because immediate treatment is critical to survival, it is desirable to have a lightweight and rapidly applicable bandage-one capable of delivering a hemostat that can quickly resolve bleeding while addressing infection over short and longer time frames. It is challenging to design thin film coatings capable of multidrug release, particularly when the drugs are quite different in nature (biologic versus small molecule, charged versus neutral) and the desired release profiles are different for each drug. Herein we have adopted a layer-by-layer film assembly technique to create a linear combination of two independently functional films capable of rapidly releasing thrombin within minutes while sustaining vancomycin elution for more than 24 h. By conjugating vancomycin to a hydrolytically degradable polyacid, poly(ß-L-malic acid), we were able to create a robust thin film with loading and release kinetics that remain unaffected by the additional deposition of a thrombin-based film, demonstrating the possibility for future multitherapeutic films with independently tunable release kinetics.

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