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1.
ACS Biomater Sci Eng ; 10(3): 1589-1606, 2024 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-38336625

RESUMO

Low molecular weight polyethylenimine (PEI) based lipopolymers become an attractive strategy to construct nonviral therapeutic carriers with promising transfection efficiency and minimal toxicity. Herein, this paper presents the design and synthesis of novel farnesol (Far) conjugated PEI, namely PEI1.2k-SA-Far7. The polymers had quick DNA complexation, effective DNA unpacking (dissociation), and cellular uptake abilities when complexed with plasmid DNA. However, they were unable to provide robust transfection in culture, indicating inability of Far grafting to improve the transfection efficacy significantly. To overcome this limitation, the commercially available polyanionic Trans-Booster additive, which is capable of displaying electrostatic interaction with PEI1.2k-SA-Far7, has been used to enhance the uptake of pDNA polyplexes and transgene expression. pDNA condensation was successfully achieved in the presence of the Trans-Booster with more stable polyplexes, and in vitro transfection efficacy of the polyplexes was improved to be comparable to that obtained with an established reference reagent. The PEI1.2k-SA-Far7/pDNA/Trans-Booster ternary complex exhibited good compatibility with cells and minimal hemolysis activity. This work demonstrates the exemplary potency of using additives in polyplexes and the potential of resultant ternary complexes for effective pDNA delivery.


Assuntos
Técnicas de Transferência de Genes , Polietilenoimina , Polietilenoimina/farmacologia , Farneseno Álcool , DNA/genética , DNA/metabolismo , Transfecção
2.
J Control Release ; 367: 821-836, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38360178

RESUMO

The clinical development of tyrosine kinase inhibitors (TKI) has led to great strides in improving the survival of chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. But even the new generation TKIs are rendered futile in the face of evolving landscape of acquired mutations leading to drug resistance, necessitating the pursuit of alternative therapeutic approaches. In contrast to exploiting proteins as targets like most conventional drugs and TKIs, RNA Interference (RNAi) exerts its therapeutic action towards disease-driving aberrant genes. To realize the potential of RNAi, the major challenge is to efficiently deliver the therapeutic mediator of RNAi, small interfering RNA (siRNA) molecules. In this study, we explored the feasibility of using aliphatic lipid (linoleic acid and lauric acid)-grafted polymers (lipopolymers) for the delivery of siRNAs against the FLT3 oncogene in AML and BCR-ABL oncogene in CML. The lipopolymer delivered siRNA potently suppressed the proliferation AML and CML cells via silencing of the targeted oncogenes. In both AML and CML subcutaneous xenografts generated in NCG mice, intravenously administered lipopolymer/siRNA complexes displayed significant inhibitory effect on tumor growth. Combining siFLT3 complexes with gilteritinib allowed for reduction of effective drug dosage, longer duration of remission, and enhanced survival after relapse, compared to gilteritinib monotherapy. Anti-leukemic activity of siBCR-ABL complexes was similar in wild-type and TKI-resistant cells, and therapeutic efficacy was confirmed in vivo through prolonged survival of the NCG hosts systemically implanted with TKI-resistant cells. These results demonstrate the preclinical efficacy of lipopolymer facilitated siRNA delivery, providing a novel therapeutic platform for myeloid leukemias.


Assuntos
Compostos de Anilina , Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide Aguda , Pirazinas , Humanos , Animais , Camundongos , RNA Interferente Pequeno , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Oncogenes , Modelos Animais , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Resistencia a Medicamentos Antineoplásicos
3.
ACS Appl Bio Mater ; 6(3): 1105-1121, 2023 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-36853230

RESUMO

Cationic polyethylenimine (PEI)-based nonviral gene carriers have been desirable to overcome the limitations of viral vectors in gene therapy. A range of PEI derivatives were designed, synthesized, and evaluated for nonviral delivery applications of plasmid DNA (pDNA). Linolenic acid, lauric acid, and oleic acid were covalently conjugated with low-molecular-weight PEI (Mw ∼ 1200 Da) via two different linkers, gallic acid (GA) and p-hydroxybenzoic acid (PHPA), that allows a differential loading of lipids per modified amine (3 vs 1, respectively). 1H NMR spectrum confirmed the expected structure of the conjugates as well as the level of lipid substitution. SYBR Green binding assay performed to investigate the 50% binding concentration (BC50) of lipophilic polymers to pDNA revealed increased BC50 with an increased level of lipid substitution. The particle analysis determined that GA- and PHPA-modified lipopolymers gave pDNA complexes with ∼300 and ∼100 nm in size, respectively. At the polymer/pDNA ratio of 5.0, the ζ-potentials of the complexes were negative (-6.55 to -10.6 mV) unlike the complexes with the native PEI (+11.2 mV). The transfection experiments indicated that the prepared lipopolymers showed higher transfection in attachment-dependent cells than in suspension cells based on the expression of the reporter green fluorescent protein (GFP) gene. When loaded with Cy3-labeled pDNA, the lipopolymers exhibited effective cellular uptake in attachment-dependent cells while the cellular uptake was limited in suspension cells. These results demonstrate the potential of lipid-conjugated PEI via GA and PHPA linkers, which are promising for the modification of anchorage-dependent cells.


Assuntos
Nanopartículas , Polietilenoimina , Polietilenoimina/química , Transfecção , DNA/química , Nanopartículas/química , Terapia Genética , Lipídeos
4.
Invest New Drugs ; 40(5): 962-976, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35834040

RESUMO

This study aimed at investigating the influence of commercial transfection reagents (Prime-Fect, Leu-Fect A, and Leu-Fect C) complexed with different siRNAs (CDC20, HSP90, Mcl-1 and Survivin) in MDA-MB-436 breast cancer cells and the impact of incorporating an anionic additive, Trans-Booster, into siRNA formulations for improving in vitro gene silencing and delivery efficiency. Gene silencing was quantitatively analyzed by real-time RT-PCR while cell proliferation and siRNA uptake were evaluated by the MTT assay and flow cytometry, respectively. Amongst the investigated siRNAs and transfection reagents, Mcl-1/Prime-Fect complexes showed the highest inhibition of cell viability and the most effective siRNA delivery. The effect of various formulations on transfection efficiency showed that the additive with 1:1 ratio with siRNA was optimal achieving the lowest cell viability compared to untreated cells and negative control siRNA treatment (p < 0.05). Furthermore, the combination of Mcl-1 and survivin siRNA suppressed the growth of MDA-MB-436 cells more effectively than treatment with the single siRNAs and resulted in cell viability as low as ~ 20% (vs. non-treated cells). This aligned well with the induction of apoptosis as analyzed by flow cytometry, which revealed higher apoptotic cells with the combination treatment group. We conclude that commercial transfection reagents formulated with Mcl-1/Survivin siRNA combination could serve as a potent anti-proliferation agent in the treatment of breast cancers.


Assuntos
Neoplasias da Mama , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Inativação Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , RNA Interferente Pequeno/genética , Survivina/genética , Survivina/farmacologia , Transfecção
5.
PLoS One ; 16(6): e0251719, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34157051

RESUMO

Overexpression and persistent activation of STAT5 play an important role in the development and progression of acute lymphoblastic leukemia (ALL), the most common pediatric cancer. Small interfering RNA (siRNA)-mediated downregulation of STAT5 represents a promising therapeutic approach for ALL to overcome the limitations of current treatment modalities such as high relapse rates and poor prognosis. However, to effectively transport siRNA molecules to target cells, development of potent carriers is of utmost importance to surpass hurdles of delivery. In this study, we investigated the use of lipopolymers as non-viral delivery systems derived from low molecular weight polyethylenimines (PEI) substituted with lauric acid (Lau), linoleic acid (LA) and stearic acid (StA) to deliver siRNA molecules to ALL cell lines and primary samples. Among the lipid-substituted polymers explored, Lau- and LA-substituted PEI displayed excellent siRNA delivery to SUP-B15 and RS4;11 cells. STAT5A gene expression was downregulated (36-92%) in SUP-B15 and (32%) in RS4;11 cells using the polymeric delivery systems, which consequently reduced cell growth and inhibited the formation of colonies in ALL cells. With regard to ALL primary cells, siRNA-mediated STAT5A gene silencing was observed in four of eight patient cells using our leading polymeric delivery system, 1.2PEI-Lau8, accompanied by the significant reduction in colony formation in three of eight patients. In both BCR-ABL positive and negative groups, three of five patients demonstrated marked cell growth inhibition in both MTT and trypan blue exclusion assays using 1.2PEI-Lau8/siRNA complexes in comparison with their control siRNA groups. Three patient samples did not show any positive results with our delivery systems. Differential therapeutic responses to siRNA therapy observed in different patients could result from variable genetic profiles and patient-to-patient variability in delivery. This study supports the potential of siRNA therapy and the designed lipopolymers as a delivery system in ALL therapy.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Portadores de Fármacos/administração & dosagem , Polímeros/administração & dosagem , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Fator de Transcrição STAT5/genética , Proteínas Supressoras de Tumor/genética , Linfócitos B/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/genética , Inativação Gênica/efeitos dos fármacos , Humanos , Ácido Linoleico/administração & dosagem , Polietilenoimina/administração & dosagem , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/genética
6.
Biomater Biosyst ; 3: 100021, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36824309

RESUMO

Uncontrolled proliferation of the myeloid cells due to BCR-ABL fusion has been successfully treated with tyrosine kinase inhibitors (TKIs), which improved the survival rate of Chronic Myeloid Leukemia (CML) patients. However, due to interactions of CML cells with bone marrow microenvironment, sub-populations of CML cells could become resistant to TKI treatment. Since integrins are major cell surface molecules involved in such interactions, the potential of silencing integrin-ß1 on CML cell line K562 cells was explored using short interfering RNA (siRNA) delivered through lipid-modified polyethyleneimine (PEI) polymers. Reduction of integrin-ß1 in K562 cells decreased cell adhesion towards human bone marrow stromal cells and to fibronectin, a major extracellular matrix protein for which integrin-ß1 is a primary receptor. Interaction of K562 cells with fibronectin decreased the sensitivity of the cells to BCR-ABL siRNA treatment, but a combinational treatment with integrin-ß1 and BCR-ABL siRNAs significantly reduced colony forming ability of the cells. Moreover, integrin-ß1 silencing enhanced the detachment of K562 cells from hBMSC samples (2 out of 4 samples), which could make them more susceptible to TKIs. Therefore, the polymeric-siRNA delivery targeting integrin-ß1 could be beneficial to reduce interactions with bone marrow microenvironment, aiding in the response of CML cells to therapeutic treatment.

7.
Pharm Res ; 37(3): 46, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32016611

RESUMO

PURPOSE: Short interfering RNA (siRNA) therapy promises a new era in treatment of breast cancers but effective delivery systems are needed for clinical use. Since silencing complementary targets may offer improved efficacy, this study was undertaken to identify non-viral carriers for combinatorial siRNA delivery for more effective therapy. METHODS: A library of lipid-substituted polymers from low molecular weight polyethyleneimine (PEI), linoleic acid (LA) and α-linoleic acid (αLA) with amide or thioester linkages was prepared and investigated for delivering Mcl-1, survivin and STAT5A siRNAs in breast cancer cells. RESULTS: The effective polymers formed 80-190 nm particles with similar zeta-potentials, but the serum stability was greater for complexes formed with amide-linked lipid conjugates. The LA and αLA substitutions, with the low molecular weight PEI (1.2 kDa and 2.0 kDa) were able to deliver siRNA effectively to cells and retarded the growth of breast cancer cells. The amide-linked lipid substituents showed higher cellular delivery of siRNA as compared to thioester linkages. Upon combinational delivery of siRNAs, growth of MCF-7 cells was inhibited to a greater extent with 2.0PEI-LA9 mediated delivery of Mcl-1 combined survivin siRNAs as compared to individual siRNAs. The qRT-PCR analysis confirmed the decrease in mRNA levels of target genes with specific siRNAs and 2.0PEI-LA9 was the most effective polymer for delivering siRNAs (either single or in combination). CONCLUSIONS: This study yielded effective siRNA carriers for combinational delivery of siRNAs. Careful choice of siRNA combinations will be critical since targeting individual genes might alter the expression of other critical mediators.


Assuntos
Neoplasias da Mama/metabolismo , Portadores de Fármacos/química , Marcação de Genes/métodos , Polietilenoimina/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Ácido Linoleico , Lipídeos , Células MCF-7 , Proteína de Sequência 1 de Leucemia de Células Mieloides , Polietilenoimina/metabolismo , Polímeros/química , Polímeros/metabolismo , Fator de Transcrição STAT5/metabolismo , Survivina/metabolismo , Proteínas Supressoras de Tumor/metabolismo
8.
J Control Release ; 310: 141-154, 2019 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-31430499

RESUMO

Despite development of effective tyrosine kinase inhibitors for treatment of chronic myeloid leukemia (CML), some patients do not effectively respond to the therapy and can display resistance in response to the drug therapy. To develop an alternative approach to CML therapy, we are exploring siRNA mediated silencing of the primary CML oncogene, BCR-ABL, by using non-viral (polymeric) delivery systems. In this study, a group of lipopolymers derived from low molecular PEIs substituted with linoleic acid (LA), α-linolenic acid (αLA) and cholesterol (Chol) was investigated for the first time for siRNA delivery to CML primary samples. The delivery efficiency in primary cells was equivalent to CML K562 cell line, and the lipopolymers gave effective internalization of siRNA depending on the nature of lipid substituent. The PEI-αLA (2.5 αLA/PEI), PEI-Chol (2.2 Chol/PEI), and PEI-LA (2.6 LA/PEI) lipopolymers used as BCR-ABL siRNA carriers (at 60 nM siRNA) reduced the BCR-ABL mRNA expression by 17% to 45%, and inhibited the formation of colonies by 24% to 41% in comparison with control siRNA in mononuclear cells. BCR-ABL siRNA treatment reduced the BCR-ABL mRNA expression by 50% in one of two CD34+ samples tested, and combination of BCR-ABL siRNA with imatinib (IM) treatment decreased the colony formation by 65% in one of two samples evaluated. The fact that no single polymer was universally effective in all patient samples may suggest patient-to-patient variability in terms of therapeutic responses to siRNA therapy. These results showed that a low dose of BCR-ABL siRNA could be used with lipopolymers to reduce BCR-ABL mRNA expression, CML cell survival and colony formation. This proof of principle study in CML primary cells can be applied to silencing of other therapeutic targets besides BCR-ABL and a study with larger patient samples is warranted for better identification of effective siRNA carriers.


Assuntos
Portadores de Fármacos/química , Proteínas de Fusão bcr-abl/genética , Inativação Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Lipídeos/química , Polietilenoimina/química , RNA Interferente Pequeno , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Colesterol/química , Feminino , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Microscopia Confocal , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ácido alfa-Linolênico/química
9.
Macromol Biosci ; 17(6)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28160423

RESUMO

Cell surface integrins, which play important roles in the survival, proliferation, migration, and invasion of cancer cells, are a viable target for treatment of metastatic breast cancer. This line of therapy still remains challenging due to the lack of proper identification and validation of effective targets as well as the lack of suitable therapeutic agents for treatment. The focus is on one such molecular target for this purpose, namely integrin-ß1, and effective lowering of integrin-ß1 levels on a breast cancer model (MDA-MB-231 cells) is achieved by delivering a dicer-substrate short interfering RNA (siRNA) targeting integrin-ß1 with lipid-modified low molecular weight polyethylenimine polymers. Reduction of integrin-ß1 levels leads to reduced adhesion of MDA-MB-231 cells to extracellular matrix component fibronectin as well as to human bone marrow cells. A reduced migration of the breast cancer cells is also observed after integrin-ß1 silencing in "scratch" and "transwell" migration assays. These results highlight the importance of integrin-ß1 for the migration of metastatic breast cancer cells by effectively silencing this target with a practical dose of siRNA.


Assuntos
Neoplasias da Mama/terapia , Técnicas de Transferência de Genes , Integrina beta1/genética , RNA Interferente Pequeno/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Inativação Gênica , Humanos , Metástase Neoplásica , Polietilenoimina/química , Polietilenoimina/uso terapêutico , RNA Interferente Pequeno/uso terapêutico
10.
Arch Oral Biol ; 70: 73-78, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27341458

RESUMO

OBJECTIVE: The objective of this in vitro study was to evaluate the effect of low-intensity pulsed ultrasound on the resorption activity of osteoclast cell cultures. DESIGN: RAW 264.7 cells were cultured and seeded over plates that were pre-coated with a synthetic carbonate apatite, and marked with fluoresceinamine-labeled sodium chondroitin polysulfate. Plates were randomly divided into 4 groups according to the treatment assigned to each one of them: NO RANKL (no RANK-L addition and no ultrasound application), NO LIPUS (addition of RANK-L and no ultrasound application), LIPUS 10 (addition of RANK-L and 10min of ultrasound application per day), and LIPUS 20 (addition of RANK-L and 20min of ultrasound application per day). The ultrasound device produced 1.5MHz pulses with a repetition rate of 1kHz and intensity of 30mW/cm2. The experiment extended for one week and afterwards, resorption activity was evaluated according to the fluorescence intensity analysis and pit resorption measurements (number of pits and mean area). RESULTS: Our experiment consistently demonstrated that low-intensity pulsed ultrasound application enhanced osteoclasts resorptive activity. In addition, it was demonstrated that when daily ultrasound application lasted longer (20min) the resorption was the highest. Results obtained from both evaluation methods were reasonably coherent. CONCLUSIONS: Low-intensity pulsed ultrasound increases osteoclast resorptive activity in the absence of osteoblasts. This effect seems to be influenced by ultrasound treatment time. Future research might be directed to investigate osteoclast response to different ultrasound application protocols (frequencies and intensities) and potential cellular mechanisms.


Assuntos
Osteoclastos/efeitos da radiação , Animais , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos da radiação , Células Cultivadas , Fluorescência , Camundongos , Ortodontia , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Ligante RANK/farmacologia , Células RAW 264.7 , Técnicas de Movimentação Dentária , Ondas Ultrassônicas
11.
J Control Release ; 224: 8-21, 2016 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-26742943

RESUMO

In spite of high complete remission rates in Acute Myeloid Leukemia (AML), little progress has been made in the long-term survival of relapsing AML patients, urging for the development of novel therapies. The CXCR4/SDF-1 axis is a potential therapeutic target in AML to reduce the enhanced survival and proliferation of leukemic cells, with current drug development efforts focusing on antagonists and blocking antibodies. The RNAi technology mediated by siRNA is a promising alternative; however, further development of clinically relevant siRNA carriers is needed since siRNA on its own is an incompetent silencing agent. Here, we report on lipid-substituted polymeric carriers for siRNA delivery to AML cells, specifically targeting CXCR4. Our results demonstrate an effective suppression of CXCR4 protein with the polymeric siRNA delivery in AML THP-1 cells. The suppression of CXCR4 as well as its ligand, SDF-1 (CXCL12), decreased THP-1 cell numbers due to reduced cell proliferation. The reduced proliferation was also observed in the presence of human bone marrow stromal cells (hBMSC), suggesting that our approach would be effective in the protective bone marrow microenvironment. The combination of CXCR4 silencing and cytarabine treatment resulted in more effective cytotoxicity when the cells were co-incubated with hBMSC. We observed a decrease in the toxicity of the lipopolymer/siRNA complexes when THP-1 cells were treated in the presence of hBMSC but this effect did not negatively affect CXCR4 silencing. In addition, siRNA delivery to mononuclear cells derived from AML patients led to significant CXCR4 silencing in 2 out of 5 samples, providing a proof-of-concept for clinical translation. We conclude that decreasing CXCR4 expression via lipopolymer/siRNA complexes is a promising option for AML therapy and could provide an effective alternative to current CXCR4 inhibition strategies.


Assuntos
Quimiocina CXCL12/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Receptores CXCR4/efeitos dos fármacos , Antimetabólitos/administração & dosagem , Antimetabólitos/uso terapêutico , Células da Medula Óssea , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Quimiocina CXCL12/genética , Citarabina/administração & dosagem , Citarabina/uso terapêutico , Sistemas de Liberação de Medicamentos , Inativação Gênica , Humanos , Lipídeos/química , Polímeros , Receptores CXCR4/genética , Células Estromais
12.
Artigo em Inglês | MEDLINE | ID: mdl-25763370

RESUMO

The cell cycle proteins are key regulators of cell cycle progression whose deregulation is one of the causes of breast cancer. RNA interference (RNAi) is an endogenous mechanism to regulate gene expression and it could serve as the basis of regulating aberrant proteins including cell cycle proteins. Since the delivery of small interfering RNA (siRNA) is a main barrier for implementation of RNAi therapy, we explored the potential of a non-viral delivery system, 2.0 kDa polyethylenimines substituted with linoleic acid and caprylic acid, for this purpose. Using a library of siRNAs against cell cycle proteins, we identified cell division cycle protein 20 (CDC20), a recombinase RAD51, and serine-threonine protein kinase CHEK1 as effective targets for breast cancer therapy, and demonstrated their therapeutic potential in breast cancer MDA-MB-435, MDA-MB-231, and MCF7 cells with respect to another well-studied cell cycle protein, kinesin spindle protein. We also explored the efficacy of dicer-substrate siRNA (DsiRNA) against CDC20, RAD51, and CHEK1, where a particular DsiRNA against CDC20 showed an exceptionally high inhibition of cell growth in vitro. There was no apparent effect of silencing selected cell cycle proteins on the potency of the chemotherapy drug doxorubicin. The efficacy of DsiRNA against CDC20 was subsequently assessed in a xenograft model, which indicated a reduced tumor growth as a result of CDC20 DsiRNA therapy. The presented study highlighted specific cell cycle protein targets critical for breast cancer therapy, and provided a polymeric delivery system for their effective down-regulation.

13.
J Mater Chem B ; 3(19): 3972-3982, 2015 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32262619

RESUMO

It has been challenging to modify primary cells with non-viral gene delivery. Herein, we developed a ternary nano-formulation for gene delivery to umbilical cord blood and bone marrow derived mesenchymal stem cells (MSC) by using lipid-modified small (1.2 kDa) molecular weight polyethylenimine (PEI1.2). Linoleic acid (LA) was end-capped with carboxyl functionality by coupling with mercaptopropionic acid through thio-ester linkage, and then grafted onto PEI1.2 via N-acylation. The thio-ester LA grafted PEI1.2 (PEI-tLA) displayed a significantly lower (up to 6-fold) DNA binding capability and a higher propensity to dissociate upon polyanionic challenge. The dissociation ability of the complexes was further enhanced by incorporating hyaluronic acid (HA) into plasmid DNA (pDNA) complexes of PEI-tLA. The HA incorporation influenced the surface charge of complexes more so than the hydrodynamic size, but it clearly increased the propensity for dissociation upon a polyanionic challenge. The PEI-tLAs were less toxic on MSC and displayed significantly higher transgene expression in MSC than conventional PEI-LA. Ternary complexes of with HA (pDNA/HA = 2, w/w) further enhanced the efficiency of PEI-tLAs of low (∼2 lipid/PEI) lipid substitution, which was comparable to or higher than commercial transfection reagents. We conclude that PEI-tLA of low lipid substitution can be employed as a gene carrier to design supersensitive nano-formulations.

14.
Leuk Res ; 38(11): 1299-308, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25262448

RESUMO

The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem/progenitor cells (LSPC) within the bone marrow (BM) niche, as well as in the high relapse rates of acute myeloid leukemia (AML). Down-regulating CD44 could be clinically relevant not only for suppression of the deregulated function of LSPC but also in LSPC response to chemotherapeutic agents. Small interfering RNA (siRNA) delivery is a promising approach for AML treatment, and we recently reported effective siRNA delivery into difficult-to-transfect AML cell lines using lipid-substituted polyethylenimine/siRNA complexes (polymeric nanoparticles). In this study, we investigated polymeric nanoparticle-mediated silencing of CD44 in CD34+ LSPC cell models (leukemic KG-1 and KG-1a cell lines) as well as primary AML cells. Polymeric nanoparticle-mediated silencing decreased surface CD44 levels in KG-1, KG-1a and primary AML cells by up to 27%, 30% and 20% at day 3, respectively. Moreover, CD44 silencing resulted in induction of apoptosis in KG-1 cells, reduced adhesion of KG-1 and KG-1a cells to hyaluronic acid-coated cell culture plates and BM-MSC, and decreased adhesion of primary AML cells to BM-MSC. Our results suggest that polymeric nanoparticle-mediated silencing of CD44 might be a useful technique for inhibiting LSPC interactions with their microenvironment, thereby prohibiting leukemia progression or sensitizing LSPC to chemotherapy.


Assuntos
Antígenos CD34/imunologia , Inativação Gênica , Receptores de Hialuronatos/metabolismo , Leucemia Mieloide Aguda/imunologia , Nanopartículas , Polímeros/química , Receptores de Antígenos/genética , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/patologia , Reação em Cadeia da Polimerase , RNA Interferente Pequeno
15.
Ann Biomed Eng ; 42(7): 1406-12, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24752635

RESUMO

Dental pulp loss due to caries or pulpitis can affect the longevity of teeth. Dental pulp tissue engineering necessitates the use of progenitor cells that has the potential to differentiate into neural, vascular and odontoblasts like cells. Previous reports have shown that human gingival progenitor cells (HGPCs) can be differentiated into different cell types; however neural differentiation of these cells, to the best of our knowledge, has not been reported. Low intensity pulsed ultrasound (LIPUS) has been reported to enhance cell differentiation. The aims of this study were (1) to explore the potential neural differentiation of HGPCs and (2) to investigate the effect of LIPUS on the differentiation of HGPCs when incubated under neuroinductive conditions. The HGPCs were isolated from human interdental papilla proximal to the premolar teeth that were extracted for orthodontic purpose. The HGPCs were induced to differentiate into neural lineage using a neuroinductive culture medium. HGPCs were divided into four groups; control group, neuro-induction (NI) group, ultrasound group (LIPUS), and a combined NI+LIPUS group. HGPCs were harvested for immunostaining and q-PCR after 1 day. Immunostaining for neuron specific antigens and q-PCR suggested that HGPCs can be differentiated into neural lineage and that selected neurodifferentiation markers can be enhanced by LIPUS.


Assuntos
Antígenos de Diferenciação/biossíntese , Diferenciação Celular , Gengiva/metabolismo , Neurônios/metabolismo , Som , Células-Tronco/metabolismo , Adolescente , Gengiva/citologia , Humanos , Masculino , Neurônios/citologia , Células-Tronco/citologia
16.
Biomater Sci ; 2(6): 833-42, 2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-26827752

RESUMO

Locally delivered plasmid DNA (pDNA) is currently pursued as gene-based therapy for regenerative medicine, but important information on in situ pDNA pharmacokinetics and transgene expression is lacking in animal models. To investigate pDNA pharmacokinetics in implants, low molecular weight (2 kDa) polyethylenimine (PEI) and linoleic acid substituted 2 kDa PEI (PEI-LA) were used for pDNA delivery in gelatin sponges. An efficient pDNA extraction method combined with quantitative PCR (qPCR) was found to give equivalent quantitation of naked and polymer-bound pDNA, making it suitable to assess pDNA polyplexes in implants. Naked pDNA implanted in a rat subcutaneous model was >98% lost after 24 hours whereas PEI and PEI-LA delivered pDNA remained intact in implants for 2 and 4 weeks, respectively. Using a plasmid expressing DsRed as a reporter gene, mRNA and protein expression was observed only for PEI-LA despite the extended retention and cellular uptake of PEI complexes. The in vivo data were in agreement with in vitro results showing that only PEI-LA was an effective transfection agent even though both PEI and PEI-LA complexes were internalized by the cells. Dose dependence was observed for mRNA expression, with a 20 µg dose giving faster onset and higher expression levels compared to a 5 µg pDNA dose. The mRNA expression after PEI-LA mediated delivery was sustained for at least 4 weeks and a significant correlation between pDNA retention in sponges and mRNA expression was observed. In addition to establishing a promising gene carrier for gene delivery, these studies provided important information about the retention and transgene expression by implanted non-viral carriers.

17.
J Control Release ; 172(1): 219-228, 2013 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23994345

RESUMO

Chemotherapy is an effective approach to curb uncontrolled proliferation of malignant cells. However, most drugs rapidly lose their efficacy as a result of resistance development. We explored the potential of combinational siRNA silencing to prevent growth of drug-resistant breast cancer cells independent of chemotherapy. Resistance was induced in two breast cancer lines by chronic exposure to doxorubicin. Microarray analysis of apoptosis-related proteins showed Bcl2, survivin, NF B, and Mcl1 to be prominently up-regulated in drug-resistant cells. Human siRNA libraries against apoptosis-related proteins and kinases were screened using lipid-substituted polymers as non-viral carrier, and siRNAs were selected to diminish cell growth without affecting growth of skin fibroblasts. Surprisingly, the selected siRNAs led to similar responses in wild-type and drug-resistant cells, despite their phenotypic differences. Promising kinase siRNAs were co-delivered with anti-apoptotic Mcl-1 siRNA and Ribosomal Protein S6 Kinase (RPS6KA5) was found the most promising candidate for simultaneous silencing with Mcl-1. In both MDA435 wild type (WT) and MDA435 resistant (R) xenografts in nude mice, double silencing of Mcl-1/RPS6KA5 also led to improved inhibition of tumor growth in the absence of chemotherapy. We conclude that combinational silencing of well-selected targets could be a feasible therapeutic strategy in the absence of drug therapy and could provide a new avenue for therapy of drug-resistant breast cancers.


Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Doxorrubicina/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Terapia Combinada , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , RNA Interferente Pequeno/genética , Proteínas Quinases S6 Ribossômicas/genética
18.
Biomaterials ; 33(11): 3363-74, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22289263

RESUMO

Non-viral delivery of genes involved in stimulation of bone formation has been pursued for clinical bone repair, but no effort has been made to assess protein expression levels after in vivo delivery. This is critical to better understand gene delivery efficiencies and to compare different modes of non-viral delivery. This study investigated expression levels of basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) after delivering expression vectors (plasmid DNA) with polymeric carriers in a rat subcutaneous implant model. The polymers used were a 2 kDa molecular weight polyethylenimine modified with linoleic acid (PEI-LA) and the 25 kDa PEI (PEI25) used for non-viral gene delivery in animal models. The PEI-LA mediated delivery of the plasmid DNAs in 293T cells led to ∼3.5 and ∼13 ng/10(6) cells/day secretion of bFGF and BMP-2 in vitro, respectively. Using the reporter protein, Green Fluorescence Protein (GFP), transfection in implants was readily detected by the presence of GFP-positive cells and a polymeric carrier was needed for this GFP expression. No bFGF and BMP-2 were detected in the scaffolds due to high background in detection assays and/or rapid diffusion of the secreted proteins from the implant site. However, using an ex vivo culture system, significant levels of BMP-2, but not bFGF, secretion were observed from the scaffolds. The BMP-2 secretion from PEI-LA delivered expression vector was equivalent and/or superior to PEI25 depending on the plasmid DNA implant dose. Gelatin scaffolds were able to sustain ∼0.3 ng/sponge/day BMP-2 secretion as compared to collagen scaffolds (∼0.1 ng/sponge/day). These values were equivalent to secretion rates reported with some viral delivery systems from independent studies.


Assuntos
Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Nanocápsulas/química , Plasmídeos/genética , Receptores de Fatores de Crescimento de Fibroblastos/biossíntese , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transfecção/métodos , Animais , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica , Ratos , Ratos Sprague-Dawley , Regulação para Cima/genética
19.
J Biomed Mater Res A ; 100(3): 684-93, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22213565

RESUMO

A conjugate of distearoylphosphoethanolamine-polyethylene glycol with 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was synthesized and incorporated into micelles and liposomes to create mineral-binding nanocarriers for therapeutic agents. The micelles and liposomes were used to encapsulate the anticancer drug doxorubicin (DOX) and a model protein lysozyme (LYZ) by using lipid film hydration (LFH) and reverse-phase evaporation vesicle (REV) methods. The results indicated that the micelles and LFH-derived liposomes were better at DOX loading than the REV-derived liposomes, while the REV method was preferable for encapsulating LYZ. The affinity of the micellar and liposomal formulations to hydroxyapatite (HA) was assessed in vitro, and the results indicated that all the thiolBP-incorporated nanocarriers had stronger HA affinity than their counterparts without thiolBP. The thiolBP-decorated liposomes also displayed a strong binding to a collagen/HA composite scaffold in vitro. More importantly, thiolBP-decorated liposomes gave increased retention in the collagen/HA scaffolds after subcutaneously implantation in rats. The designed liposomes were able to entrap the bone morphogenetic protein-2 in a bioactive form, indicating that the proposed nanocarriers could deliver bioactive factors locally in mineralized scaffolds for bone tissue engineering.


Assuntos
Conservadores da Densidade Óssea , Doenças Ósseas/tratamento farmacológico , Difosfonatos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Lipídeos/química , Nanopartículas/química , Animais , Materiais Biocompatíveis/química , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/uso terapêutico , Osso e Ossos/metabolismo , Difosfonatos/química , Difosfonatos/uso terapêutico , Feminino , Lipossomos/química , Teste de Materiais , Micelas , Estrutura Molecular , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley
20.
J Drug Target ; 18(8): 611-26, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20158316

RESUMO

A polymeric conjugate of polyethyleneimine-graft-poly(ethylene glycol) and 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (PEI-PEG-thiolBP) was prepared and used for surface coating of bovine serum albumin (BSA) nanoparticles (NPs) designed for bone-specific delivery of bone morphogenetic protein-2 (BMP-2). The NP coating was achieved with a dialysis and an evaporation method, and the obtained NPs were characterized by particle size, zeta-potential, morphology, and cytotoxicity in vitro. The particle size and surface charge of the NPs could be effectively tuned by the PEG and thiolBP substitution ratios of the conjugate, the coating method, and the polymer concentration used for coating. The PEG modification on PEI reduced the toxicity of PEI and the coated NPs, based on in vitro assessment with human C2C12 cells and rat bone marrow stromal cells. On the basis of an alkaline phosphatase (ALP) induction assay, the NP-encapsulated BMP-2 displayed full retention of its bioactivity, except for BMP-2 in PEI-coated NPs. By encapsulating (125)I-labeled BMP-2, the polymer-coated NPs were assessed for hydroxyapatite (HA) affinity; all NP-encapsulated BMP-2 showed significant affinity to HA as compared with free BMP-2 in vitro, and the PEI-PEG-thiolBP coated NPs improved the in vivo retention of BMP-2 compared with uncoated NPs. However, the biodistribution of NPs after intravenous injection in a rat model indicated no beneficial effects of thiolBP-coated NPs for bone targeting. Our results suggested that the BP-conjugated NPs are useful for localized delivery of BMP-2 in bone repair and regeneration, but they are not effective for bone targeting after intravenous administration.


Assuntos
Proteína Morfogenética Óssea 2/administração & dosagem , Osso e Ossos/metabolismo , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Soroalbumina Bovina , Animais , Proteína Morfogenética Óssea 2/farmacocinética , Bovinos , Linhagem Celular , Difosfonatos/química , Portadores de Fármacos , Durapatita/metabolismo , Excipientes , Humanos , Nanopartículas/análise , Nanopartículas/toxicidade , Tamanho da Partícula , Polietilenoglicóis/química , Polietilenoimina/análogos & derivados , Polietilenoimina/química , Ratos , Propriedades de Superfície , Distribuição Tecidual
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