Cholesterol grafted cationic lipopolymers: Potential siRNA carriers for selective chronic myeloid leukemia therapy.

Synthetic siRNA technology has emerged as a promising approach for molecular therapy of cancer but, despite its potential for post-transcriptional gene silencing, there is an urgent need to develop efficient delivery systems particularly for difficult-to-transfect, anchorage-independent cells. In this study, we designed highly hydrophobic cationic lipopolymers by grafting cholesterol (Chol) onto low-molecular weight (0.6, 1.2, and 2.0 kDa) polyethylenimines (PEIs) to enable specific siRNA therapy to chronic myeloid leukemia (CML) cells. The siRNA binding by PEI-Chol led to nano-sized (100-200 nm diameter) polyplexes with enhanced ζ-potential (+20 to +35 mV) and ability to protect the loaded siRNA completely in fresh serum. The siRNA delivery to CML (K562) cells was proportional to degree of substitution and, unexpectedly, inversely proportional to molecular size of the polymeric backbone. Chol grafting with as little as ~1.0 Chol/PEI on 0.6 and 1.2 kDa PEIs enabled silencing of the reporter Green Fluorescent Protein gene as well as the endogenous BCR-Abl oncogene in K562 cells. The PEI-Chol mediated delivery of siRNAs specific for BCR-Abl and KSP genes significantly arrested the growth the cells which was significantly reflected in colony formation potency of K562 cells. BCR-Able siRNA mediated therapeutic efficacy was also observed in significantly increased caspase activity and apoptosis of K562 cells. Thus, Chol-grafted low-molecular weight PEIs appear to be unique siRNA carriers to realize the molecular therapy in CML cells.

siRNA-mediated BCR-ABL silencing in primary chronic myeloid leukemia cells using lipopolymers.

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.

Current outlook on drug resistance in chronic myeloid leukemia (CML) and potential therapeutic options.

Chronic myeloid leukemia cells are armed with several resistance mechanisms that can make current drugs ineffective. A better understanding of resistance mechanisms is yielding new approaches to management of the disease. Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm the hallmark of which, the breakpoint cluster region-Abelson (BCR-ABL) oncogene, has been the target of tyrosine kinase inhibitors (TKIs), which have significantly improved the survival of patients with CML. However, because of an increase in TKI resistance, it is becoming imperative to identify resistance mechanisms so that drug therapies can be better prescribed and new agents developed. In this review, we discuss the various BCR-ABL-dependent and -independent mechanisms of resistance observed in CML, and the range of therapeutic solutions available to overcome such resistance and to ultimately improve the survival of patients with CML.

siRNA/lipopolymer nanoparticles to arrest growth of chronic myeloid leukemia cells in vitro and in vivo.

Therapies for the treatment of Chronic Myeloid Leukemia and other leukemias are still limited for patients at advanced stages, which allow development of point mutations in the BCR-ABL fusion gene that render CML cells insensitive to therapies. An effective non-viral delivery system based on lipopolymers is described in this study to deliver specific siRNAs to CML cells for therapeutic gene silencing. The lipopolymer, based on the lipid α-linolenic acid (αLA) substitution on low molecular weight polyethyleneimine (PEI), was used to deliver siRNA against the BCR-ABL gene and, the resultant therapeutic effect was evaluated in in vitro and in vivo CML models. The study concluded that siRNA/PEI-αLA nanoparticles enabled silencing of the BCR-ABL gene and BCR-ABL protein, which consequently reduced growth on CML K562 cells in vitro and arrested the growth of localized tumors in a localized CML mouse model. The results from this study confirmed the potential use of lipopolymers as delivery systems and are encouraging for the future design of non-viral delivery systems for the treatment of CML and other hematological malignancies resulting from gene fusions.

Nucleic acid combinations: A new frontier for cancer treatment.

The emerging molecular understanding of cancer cell behavior is leading to increasing possibilities to control unchecked cell growth and metastasis. On the other hand, development of multifunctional drug carriers at the 'nano'-scale is providing exciting new therapeutic strategies in clinical management of cancer beyond the conventional cytotoxic drugs. A new frontier in this regard is the combinational use of complementary agents based on nucleic acids to overcome the limitations of conventional therapy. The existence of tightly-integrated cross-talk through multiple signaling and effector pathways have been appreciated for some time, and the plasticity of such a network to overcome one-dimensional intervention is stimulating development of combinational therapy. The objective of this review is to underline the cutting edge technologies and opportunities employed in combination cancer therapy using nucleic acids therapeutics for successful clinical translation. Here, we provide a detailed analysis of the multifunctional carriers designed for different types of payloads, surveying the biomaterials used to construct the functional carriers. We then provide effective nucleic acid combinations employed to obtain more comprehensive outcomes, highlighting the critical factors involved in successful therapy. We conclude with an authors' perspective on the future of combinational therapy using nucleic acid therapeutics, articulating the main challenges to advance this promising approach to the clinical realm.

Fibronectin-modified surfaces for evaluating the influence of cell adhesion on sensitivity of leukemic cells to siRNA nanoparticles.

AIM: This study aimed to create fibronectin (FN)-grafted polymeric surfaces to investigate the influence of leukemic cell adhesion on siRNA treatment. MATERIALS & METHODS: FN was grafted on plasma-treated PTFE surfaces using chemical crosslinkers. Adhesion and growth of chronic myeloid leukemia K562 cells on modified surfaces were investigated. The silencing effect of siRNA/lipid-polymers nanoparticles on cells grown on FN-grafted surfaces was evaluated. RESULTS: Crosslinker-mediated immobilization showed significant FN grafting on surfaces, which provided K562 cell adhesion and growth advantage. siRNA nanoparticle silencing was similarly effective on FN-adhered and suspension-growing K562 cells. CONCLUSION: This study provided initial data to develop a cell-adhesive system to investigate therapeutic effects on leukemic cells. The response of chronic myeloid leukemia cells to siRNA nanoparticles was independent on cell attachment.

Current attempts to implement siRNA-based RNAi in leukemia models.

Leukemias arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to abnormal blood population with transformed cells. With the advent of RNAi and its pharmacological mediator siRNA, it has become possible to downregulate specific drivers causing leukemias. In this review, we present unique aspects of RNAi-mediated therapy and delivery technologies. Recent updates on molecular targets and delivery systems are discussed emanating from in vitro cell models and preclinical animal models. We conclude with a view on the future of RNAi in leukemia therapy, emphasizing possible measures to achieve higher efficacy and improved safety.

Progress in RNAi-mediated Molecular Therapy of Acute and Chronic Myeloid Leukemia.

Leukemias arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to impaired regulation of proliferation, differentiation, apoptosis, and survival of the transformed cells. With the advent of RNA interference (RNAi) and the short interfering RNA (siRNA) as its pharmacological mediator, it is becoming possible to modulate specific targets at will. This article summarizes current attempts to utilize RNAi reagents for therapy of leukemias, focusing on acute and chronic myeloid leukemia. We first present unique aspects of RNAi-mediated therapy, followed by a brief background on the delivery technology of RNAi reagents. The need for leukemia-specific delivery of siRNA is discussed by describing approaches that targeted agents to leukemic cells. Pharmacokinetics and biodistribution of RNAi agents are then presented, highlighting the critical issues pertinent to emerging siRNA therapy. Efforts to deliver specific RNAi therapies are then summarized in the context of expected clinical outcomes, focusing on limiting leukemic cell survival, sensitizing malignant cells to chemotherapy, mobilization of leukemic cells, and eradication of leukemic stem cells. We conclude with a perspective on the future of RNAi therapy, emphasizing the technological requirements and mechanistic challenges for clinical entry.

Polymeric nanoparticle-mediated silencing of CD44 receptor in CD34+ acute myeloid leukemia cells.

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.

siRNA therapy in cutaneous T-cell lymphoma cells using polymeric carriers.

Cutaneous T-cell lymphomas (CTCLs) arise from specific molecular aberrations that lead to uncontrolled cell proliferation. RNA interference (RNAi) with short interfering RNAs (siRNAs) is a feasible approach to interrupt aberrant signal processing in CTCL cells, but functional biomaterial carriers are needed to effectively deliver siRNAs intracellularly. Towards this goal, we explored the utility of lipid-substituted polyethylenimines (PEI) carriers in a cell model of CTCL. Using caprylic and linoleic acid substituted 2 kDa PEI (PEI-CA and PEI-LA, respectively), we showed effective delivery of siRNA to T-lymphocyte Hut78 and Jurkat cells, but silencing of a model protein (Green Fluorescent Protein, GFP) was possible only in the Hut78 cells. To enhance siRNA delivery to Hut78 cells, a high siRNA: carrier ratio used to assemble the complexes and centrifugation of cells in the presence of complexes were found effective. The toxicities of PEI-CA and PEI-LA were significantly lower than other commercial carriers, 25 kDa PEI and Lipofectamine(®) RNAiMAX. This might have contributed to reduced siRNA delivery efficiency of the latter carriers. Screening several endogenous targets led us to identify phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and cyclin-dependent kinase 18 (CDK18) as viable targets to induce siRNA-mediated cell growth inhibition. The results of this study identified promising polymeric carriers and molecular targets that could control proliferation of CTCL cells based on RNAi therapy.

Investigating siRNA delivery to chronic myeloid leukemia K562 cells with lipophilic polymers for therapeutic BCR-ABL down-regulation.

RNAi represents a new alternative for treatment of chronic myeloid leukemia (CML) to overcome the difficulties of current drug treatments such as the acquired resistance. However, potent carriers that can overcome delivery barriers to RNAi agents and have therapeutic efficacy especially in difficult-to-transfect CML cells are needed. Here, we explored the use of lipid-modified polyethylenimines (PEI) of low molecular weights (0.6, 1.2 and 2.0kDa) in K562 cells and showed that the delivery efficiency was dependent on the type of lipid used for polymer modification, degree of lipid substitution and polymer molecular weight. Among the lipid-substituted polymers investigated, palmitic acid (PA)-substituted 1.2kDa PEI (~2 lipids/PEI) has proven to be highly efficient in delivering siRNA and silencing of the reporter gene green fluorescent protein (GFP). The silencing efficacy achieved with this polymer was found to be higher than the 25kDa PEI and is similar to commercial reagent Lipofectamine™ 2000. Moreover, when BCR-ABL protein was targeted in K562 cells, a reduction in the corresponding mRNA levels was observed, as well as an induction of early and late stage apoptosis. The results of this study demonstrated that PA-substitutions on low MW polymers could be useful for siRNA delivery in CML cells for therapeutic purposes.

CONSTRUCCIÓN DE MATRICES COMO SUSTITUTOS DÉRMICOS: APLICACIÓN POTENCIAL EN LA REGENERACIÓN DE PIEL / MATRIX CONSTRUCTION AS DERMAL SUBSTITUTES: POTENTIAL APPLICATION IN SKIN REGENERATION

La pérdida de grandes porciones de piel limita su capacidad de regeneración, haciendo difícil su tratamiento. Mediante la ingeniería de tejidos se han desarrollado sustitutos dérmicos que promueven la regeneración; sin embargo, es necesario hallar un material para el crecimiento de fibroblastos que sea un sustituto dérmico adecuado. En este trabajo se construyeron matrices tridimensionales porosas de colágeno y colágeno-ácido hialurónico evaluando su potencial como sustituto dérmico. Las matrices se entrecruzaron con 1-etil-3-(3-dimetilaminopropil) carbodiimida hidrocloruro. Se evaluó la porosidad, se realizaron pruebas de degradación, evaluación de adherencia celular con fibroblastos humanos y, ensayos de viabilidad y proliferación. Las matrices presentaron poros de 50 µm de diámetro uniformemente distribuidos. Las matrices de colágeno y colágeno-ácido hialurónico (2:8 y 4:6) presentaron una degradación progresiva con pesos similares. Luego de 24 horas en cultivo, los resultados de la prueba de MTT mostraron que los fibroblastos en colágeno proliferaron; mientras que los fibroblastos en colágeno-ácido hialurónico no evidenciaron proliferación. Aunque la estructura porosa y la tasa de degradación de las matrices son similares, las de colágeno brindan mejores condiciones de adhesión y proliferación a los fibroblastos cultivados en comparación con las que tienen ácido hialurónico como adición, convirtiéndose en el mejor andamio para ser empleado como sustituto dérmico entre las matrices evaluadas.

Wounds can cause loss of skin that limits the skin regeneration, making the treatment more difficult to address. The tissue engineering has developed skin substitutes that promote skin regeneration; however, it is imperative to find materials that allow fibroblast growth in order to find an appropriate skin substitute. The construction of tridimensional porous collagen and collagenhyaluronic acid matrixes crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride is presented in order to evaluate their potential use in skin regeneration therapies. Matrix porosity was evaluated. Degradation tests, human fibroblast adherence, viability and proliferation tests were performed. Matrixes had a uniform distributed porosity with mean diameters of 50 µm. Both collagen and collagen-hyaluronic acid (2:8 and 4:6) matrixes presented a progressive degradation rate with similar weight. After 24 hours under culture, the number of fibroblasts seeded on collagen matrixes were doubled, while the number of fibroblast seeded on collagen-hyaluronic acid matrixes remained similar. Even though porous structure and degradation rate of different types of constructed matrixes in this study are similar, collagen matrixes offer better adhesion and proliferation conditions for seeded fibroblasts in comparison with hyaluronic acid added matrixes, making them the best scaffold to be employed as a dermic substitute among the matrixes compared here.