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Basic fibroblast growth factor (bFGF) is capable of stimulating osteogenic differentiation of preosteoblast cells in vitro and new bone tissue deposition in vivo. Delivering the gene for the protein, rather than the protein itself, is considered advantageous for bone repair since gene delivery obviates the need to produce the protein in pharmaceutical quantities. To explore the feasibility of bFGF gene delivery by nonviral methods, we transfected primary rat bone marrow stromal cells (BMSC) using cationic polymers (polyethylenimine and poly(L-lysine)-palmitic acid) in vitro. After delivering a bFGF-expression plasmid (pFGF2-IRES-AcGFP) to BMSC, the presence of bFGF in culture supernatants was detected by a commercial ELISA. As much as 0.3 ng bFGF/10(6) cells/day was obtained from the BMSC under optimal conditions. This secretion rate was approximately 100-fold lower than the secretion obtained from immortal, and easy-to-transfect, human 293T cells. These data suggest the feasibility of modifying BMSC with nonviral delivery systems for bFGF expression, but also highlight the need for substantial improvement in transfection rate for an effective therapy. [ABSTRACT FROM AUTHOR]

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled. RNA interference (RNAi), mediated by short interfering RNA (siRNA), presents a mechanistically distinct therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. This study explored the use of a non-viral approach for delivery of FLT3 siRNA (siFLT3) in FLT3-ITD positive AML cell lines and primary cells as well as the feasibility of combining this treatment with drugs currently used in the clinic. Treatment of AML cell lines with FLT3 siRNA nanocomplexes resulted in prominent reduction in cell proliferation rates and induction of apoptosis. Quantitative analysis of relative mRNA transcript levels revealed downregulation of the FLT3 gene, which was accompanied by a similar decline in FLT3 protein levels. Moreover, an impact on leukemic stem cells was observed in a small pool of primary AML samples through significantly reduced colony numbers. An absence of a molecular response post-treatment with lipopolymer/siFLT3 complexes in peripheral blood mononuclear cells, obtained from healthy individuals, denoted a passive selectivity of the complexes towards malignant cells. The effect of combining lipopolymer/siFLT3 complexes with daunorubucin and FLT3 targeting TKI gilteritinib led to a significant augmentation of anti-leukemic activity. These findings demonstrate the promising potential of RNAi implemented with lipopolymer complexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML. STATEMENT OF SIGNIFICANCE: We show that a clinically validated target, the FLT3 gene, can be eradicated in leukemia cells using non-viral RNAi. We validated these lipopolymers as effective vehicles to deliver nucleic acids to leukemic cells. The potency of the lipopolymers was superior to that of the 'gold-standard' delivery agent, lipid nanoparticles (LNPs), which are not effective in leukemia cells at clinically relevant doses. Mechanistic studies were undertaken to probe structure-function relationships for effective biomaterial formulations. Cellular and molecular responses to siRNA treatment have been characterized in cell models, including leukemia patient-derived cells. The use of the siRNA therapy with clinically used chemotherapy was demonstrated., Competing Interests: Declaration of interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hasan Uludag reports financial support was provided by RJH Biosciences Inc. Hasan Uludag and Remant K.C. reports a relationship with RJH Biosciences Inc. that includes: equity or stocks and funding grants. Hasan Uludag has patent issued to RJH Biosciences. Member of editorial board, Biomaterials. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The therapeutic potential of small interfering RNAs (siRNAs) in gene-targeted treatments is substantial, but their suboptimal delivery impedes widespread clinical applications. Critical among these is the inability of siRNAs to traverse the cell membranes due to their anionic nature and high molecular weight. This limitation is particularly pronounced in lymphocytes, which pose additional barriers due to their smaller size and scant cytoplasm. Addressing this, we introduce an innovative lipid-conjugated polyethylenimine lipopolymer platform, engineered for delivery of therapeutic siRNAs into lymphocytes. This system utilizes the cationic nature of the polyethylenimine for forming stable complexes with anionic siRNAs, while the lipid component facilitates cellular entry of siRNA. The resulting lipopolymer/siRNA complexes are termed lipopolymer nanoparticles (LPNPs). We comprehensively profiled the efficacy of this platform in human peripheral blood mononuclear cells (PBMCs) as well as in vitro and in vivo models of acute lymphoblastic leukemia (ALL), emphasizing the inhibition of the oncogenic signal transducer and activator of transcription 5A ( STAT5A) gene. The lipopolymers demonstrated high efficiency in delivering siRNA to ALL cell lines (RS4;11 and SUP-B15) and primary patient cells, effectively silencing the STAT5A gene. The resultant gene silencing induced apoptosis and significantly reduced colony formation in vitro . Furthermore, in vivo studies showed a significant decrease in tumor volumes without causing substantial toxicity. The lipopolymers did not induce the secretion of proinflammatory cytokines (IL-6, TNF-α, and INF-γ) in PBMCs from healthy volunteers, underscoring their immune safety profile. Our observations indicate that LPNP-based siRNA delivery systems offer a promising therapeutic approach for ALL in terms of both safety and therapeutic efficacy., Competing Interests: The authors declare the following competing financial interest(s): M.N., A.P.R., and D.N.M.S. were supported by a fellowship from RJH Biosciences Inc. R.K.C. and H.U. have ownership interest in RJH Biosciences Inc. (Edmonton, Alberta, Canada)., (© 2024 American Chemical Society.)

Chikungunya virus is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates following the introduction of its major vector, Aedes albopictus . Recent cases have been documented in Europe, the Caribbean, and the Americas. Chikungunya virus causes a disease frequently misdiagnosed as Dengue fever, with potentially life-threatening symptoms that can result in long term debilitating arthritis. There have been ongoing investigations of possible therapeutic interventions for both acute and chronic symptoms, but to date none have proven effective in reducing the severity or lasting effects of this disease. Recently, a promising vaccine candidate has received accelerated approval, indicating the importance of remedies to this emerging worldwide health threat. Nonetheless, therapeutic interventions for Chikungunya and other mosquito borne virus diseases are urgently needed yet remain elusive. The increasing risk of spread from endemic regions via human travel and commerce, coupled with the absence of a vaccine or approved therapeutic, puts a significant proportion of the world population at risk for this disease. In this report we explore the possibility of using Specific On/oFf Adapter Hepatitis Delta Virus Ribozymes as antivirals in cells infected with Chikungunya virus. The results we obtained suggest there could be some role in using these ribozyme molecules as antiviral therapies for not only Chikungunya virus, but potentially other viruses as well.

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.

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., Competing Interests: Declaration of competing interest A.S.A., C.K., X.J., and J.B. declare no competing financial interests. L.M., M.N., and D.N. were supported by a fellowship from RJH Biosciences Inc. R.K.C. and H.U. have ownership interest in RJH Biosciences Inc. (Edmonton, Alberta, Canada)., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 ( M w ∼ 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). 1 H 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 (BC 50 ) of lipophilic polymers to pDNA revealed increased BC 50 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.

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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-β1 on CML cell line K562 cells was explored using short interfering RNA (siRNA) delivered through lipid-modified polyethyleneimine (PEI) polymers. Reduction of integrin-β1 in K562 cells decreased cell adhesion towards human bone marrow stromal cells and to fibronectin, a major extracellular matrix protein for which integrin-β1 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-β1 and BCR-ABL siRNAs significantly reduced colony forming ability of the cells. Moreover, integrin-β1 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-β1 could be beneficial to reduce interactions with bone marrow microenvironment, aiding in the response of CML cells to therapeutic treatment., Competing Interests: HU and RBKC are founders of a start-up company (RJH Biosciences) commercializing the polymers for nucleic acid delivery. Other authors declare that there are no conflicts of interest., (© 2021 The Authors. Published by Elsevier Ltd.)

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., Competing Interests: HU is a founding shareholder in RJH Biosciences Inc., which is commercializing the described transfection reagents. This does not alter our adherence to PLOS ONE policies on sharing data and materials. No other authors have any competing interests to declare.

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rationale and Objective: This study aimed to evaluate the use of an adenosine triphosphate (ATP) monitoring system to minimize surface contamination on inpatient computed tomography (CT) scanners., Methods: The bore, table, and wrap of two quaternary care inpatient CT scanners (load/scanner: ~ 30-40 CT examinations/day) were assayed with bacterial cultures and an ATP detection system during six prospective iterative plan-do-check-act improvement cycles from January 6, 2016 to October 12, 2016. Per-cycle sampling was for eight consecutive weekdays. ATP detection was expressed as relative light units (RLUs) through a luciferase reaction, with >350 RLU considered contaminated per manufacturer recommendations. Culture swabs were placed into 6.5% NaCl broth, a Staphylococcus enrichment broth, and incubated aerobically at 37°C for 48 hours. Positive broths were plated to chromogenic Staphylococcus media. Culture rates (Fisher exact test) and RLU values (Mann-Whitney U test) were compared., Results: In Cycle 1, both culture results and median RLU values indicated the wrap was the most contaminated item (positive culture rate: 63% [10/16], median RLU interquartile range: 173 [IQR: 56-640]); however, RLU values were not predictive of per-sample culture results (P = .36). Following iterative improvements, RLU values at Cycle 6 were significantly lower than at peak (P = .02-.04) and within manufacturer's recommendations: all samples: 45 (IQR: 16-87), bore: 26 (IQR: 0-51), table: 68 (IQR: 21-89), wrap: 47 (IQR: 38-121)., Conclusion: The Velcro wrap is the most contaminated item on a CT scanner, and special processes may be needed to ensure adequate cleansing. ATP detection is a crude surrogate for bacterial culture results but benefits from speed, reduced cost, and greater statistical power., (Copyright © 2017 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

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-β1, and effective lowering of integrin-β1 levels on a breast cancer model (MDA-MB-231 cells) is achieved by delivering a dicer-substrate short interfering RNA (siRNA) targeting integrin-β1 with lipid-modified low molecular weight polyethylenimine polymers. Reduction of integrin-β1 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-β1 silencing in "scratch" and "transwell" migration assays. These results highlight the importance of integrin-β1 for the migration of metastatic breast cancer cells by effectively silencing this target with a practical dose of siRNA., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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/cm 2 . 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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

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.

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., (© 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Wiley Periodicals, Inc.)

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.

Bone Morphogenetic Protein-2 (BMP-2) plays an important role in stimulating new bone formation, and has been utilized in clinical bone repair by implantation. In this study, we report a nanoparticulate (NP) system for BMP-2 delivery based on bovine serum albumin (BSA) NPs stabilized with a poly(ethylene glycol) modified polyethylenimine (PEI-PEG) coating. PEI-PEG with different PEG substitutions were synthesized, and the cell viability assay showed PEG substitution greatly reduced the cytotoxicity of the native PEI. Furthermore, PEI-PEG coated BSA NPs demonstrated smaller size and decreased zeta potential compared to PEI-coated NPs. The bioactivity of the encapsulated BMP-2 and the toxicity of PEI-PEG coated NPs were examined by the alkaline phosphatase (ALP) induction assay and the MTT assay, respectively, using human C2C12 cells. The results indicated that BMP-2 remained bioactive in NPs and PEI-PEG coating was advantageous in reducing the NP toxicity as compared to PEI. A 7-day pharmacokinetics study showed the BMP-2 retention in PEI-PEG coated NPs was similar to the uncoated NPs, but lower than that of the PEI-coated NPs. The osteoinductivity of BMP-2 delivered in NPs was determined by subcutaneous implantation in rats, and the results revealed that PEI-PEG coated BSA NPs induced significant de novo bone formation after implantation, while PEI-coated NPs demonstrated much less bone formation. We conclude that BMP-2 delivered by PEGylated PEI-coated BSA NPs displays favorable biocompatibility and promotes new bone formation after implantation.

The current study compared the effectiveness of an amphiphilic biomaterial poly(L-lysine)-palmitic acid (PLL-PA), and the lipid-based transfection agent Lipofectamine 2000 for plasmid delivery to bone marrow stromal cells (BMSC). We investigated the utility of the carriers to deliver a plasmid containing enhanced green fluorescent protein (pEGFP) to BMSC in vitro. Confocal microscopy was used to investigate the intracellular trafficking of pEGFP/carrier complexes. pEGFP delivery and EGFP expression were assessed by flow cytometry. PLL-PA formed condensed structures with pEGFP and successfully delivered the plasmid into the nucleus within 5 h of incubation with the cells. PLL-PA delivered the pEGFP to approximately 80% of the cells, achieving a maximum transfection efficiency of approximately 22%. This was significantly higher than Lipofectamine 2000-mediated transfection, which was 11% under most optimal conditions. Dosing the BMSC two or three times during the 24 h period increased the transfection efficiency by 2-3 folds, without compromising cell viability. When chloroquine was employed as an ensomolytic agent, 100 microM of the drug increased the transfection efficiency while reducing cell viability, but lower concentrations (1-10 microM) were not beneficial for transfection. Combining PLL-PA with Lipofectamine 2000 created an additive effect, increasing the transfection efficiency of PLL-PA. Long-term evaluation of gene expression with pEGFP/PLL-PA yielded approximately 17% transfection on day 1, which gradually decreased over a 12-day period. We conclude that PLL-PA is an effective biomaterial carrier and a promising candidate for non-viral gene delivery to BMSC.

Nonviral gene carriers are actively explored in gene therapy due to safety concerns of the viral carriers. To design effective gene carriers for modification of bone marrow stromal cells (BMSC), an important cell phenotype for clinical application of gene therapy, cationic polymers polyethyleneimine (PEI), and poly-L-Lysine (PLL) were substituted with palmitic acid (PA) via amide linkages. Depending on the reaction conditions, PEI and PLL was substituted with 2.2-5.2 and 13.4-16.2 PA per polymer chain. The PA substituted polymers displayed slightly lower binding efficiency towards a plasmid containing Enhanced Green Fluorescent Protein (pEGFP) in an agarose gel binding assay. The cell binding of PLL-PA, but not PEI-PA, was particularly enhanced, resulting in higher percentage of the cells displaying a significant polymer uptake. pEGFP delivery into the BMSC was also significantly increased with the PLL-PA (vs. PLL), but not PEI-PA (vs. PEI). The transfection efficiency of PLL-PA was significantly higher ( approximately fivefold) than the unmodified polymer. We conclude that PA substitution on PLL provides an effective carrier for transfection of primary cells derived from the bone marrow., (Copyright 2007 Wiley Periodicals, Inc.)

Basic fibroblast growth factor (bFGF) is a potent mitogen that exhibits stimulatory effects on bone tissue regeneration. To gain further insight into the potential of bFGF for systemic therapy in osteoporosis, we investigated the responsiveness of bone marrow stromal cells (BMSCs) explanted from 7-month-old normal and ovariectomized (OVX) rats that were intravenously treated with a low dose of bFGF (25 microg/kg) for 2 weeks. The BMSCs were obtained using femoral aspiration and maintained in an osteogenic medium. The amount of cells recovered from bFGF-treated rats was lower than that from saline-treated rats, and proliferation of the cells was markedly less for the bFGF-treated rats. The BMSCs from the bFGF-treated rats also showed lower levels of specific alkaline phosphatase (ALP) activity (ALP/deoxyribonucleic acid) and mineralization. Expression of the extracellular matrix proteins critical for mineralization, in particular osteopontin, was greater for bFGF-treated cells from both types of animals in the first week of culture, after which the expression of all markers significantly declined. Dual energy x-ray absorptiometry analyses of the tibiae showed an increase in bone mineral density after bFGF treatment only for OVX rats. We conclude that osteoprogenitor cells were depleted from the marrow of bFGF-treated rats, most likely because of the stimulatory effect of bFGF on bone formation.

Bone marrow stromal cells (BMSC) represent an important cell phenotype for pursuit of successful gene therapy. Non-viral methods to enable expression of exogenous genes in BMSC will accelerate clinical application of gene therapy, without the concerns associated with the viral means of gene transfer. Towards this end, this study investigated the potential of cationic polymers poly-L-lysine (PLL) and branched polyethylenimine (PEI) as gene carriers for modification of BMSC. Both polymers rapidly (approximately 30 min) condensed a 4.2 kb Enhanced Green Fluorescent Protein (pEGFP-N2) plasmid into 100-200 nm particles. PLL and PEI were both readily internalized with BMSC with >80% of BMSC exhibiting polymer uptake by flow cytometric analysis. The relative uptake of PEI, however, was significantly higher as compared to the PLL. The majority of the BMSC (>60%) exhibited nuclear presence of the polymers as analyzed by fluorescent microscopy. Although both polymers were able to deliver the pEGFP-N2 into the cells under microscopic evaluation, only a small fraction of the cells (<10%) displayed nuclear localization of the plasmid. Consistent with better uptake, PEI gave a higher delivery of pEGFP-N2 into the BMSC, which resulted in a more sustained expression of the model gene EGFP in short-term (7-day) culture. We conclude that both PLL and PEI readily displayed cellular uptake, but PEI was more effective in delivering plasmid DNA intracellularly, which was likely the underlying basis for a more sustained gene expression.

Bone marrow stromal cells (BMSC) modified with therapeutic genes are being actively pursued for gene therapy protocols. To develop safe and effective nonviral methods for BMSC modification, the cationic polymer polyethyleneimine (PEI) has been utilized to condense plasmid DNA for intracellular delivery. This study was conducted to explore the feasibility of increasing the PEI's effectiveness by coupling integrin-binding arginine-glycine-aspartic acid (RGD) peptides to the polymer. BMSC from rats were isolated and expanded in culture for gene transfer studies. In contrast to our expectations, RGD-conjugated PEI did not exhibit an enhanced binding to BMSC. This was the case where the peptides were conjugated to PEI by short, disulfide linkages or long poly(ethylene glycol) linkages. Using a reporter gene for the enhanced green fluorescent protein, the transfection efficiency of RGD-conjugated PEI was also lower than the delivery by the native PEI, which exhibited equivalent transfection efficiency to that of an adenovirus. We conclude that native PEI was sufficient for the transformation of BMSC and that coupling of the integrin-binding RGD-peptides did not improve the effectiveness of this polymer for BMSC transfection.

Basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) are actively pursued for stimulation of bone formation. To assess their promise for systemic therapy of osteoporosis, we ascertained the effects of bFGF and BMP-2 on bone marrow cells in vitro. Bone marrow cells were obtained from young (8 weeks) and adult (32 weeks) rats by femoral aspiration and were exposed to osteogenic medium (ie, basal medium with 10 mM beta-glycerolphosphate and 100 nM dexamethasone) containing the growth factors. The cell viability in osteogenic medium was reduced after 3 weeks but not if the concentration of beta-glycerolphosphate/dexamethasone was reduced to 3 mM/30 nM. Unlike BMP-2, bFGF at 2-50 ng/mL was capable of enhancing long-term cell viability. Continuous treatment of bone marrow cells for 3 weeks resulted in dose-dependent stimulation of mineralization by BMP-2, but not by bFGF, whose activity was optimal at 2-10 ng/mL. To explore the effect of short-term exposure, bone marrow cells were treated with growth factors for 1 week and subsequent mineralization was investigated. BMP-2 exposure increased the extent of mineralization, but bFGF was not effective after the short exposure. We concluded bFGF was more potent (ie, required lower concentration) for stimulating osteogenic parameters, but BMP-2 effects were lasting on the bone marrow cells.

Chemical conjugation of bisphosphonates (BPs) to therapeutic proteins is an effective means to impart mineral affinity to proteins. Such conjugates can be implanted with mineral-based matrices to control the local delivery kinetics of the proteins. BPs linked to proteins with reversible (i.e., cleavable) linkages are desirable over conjugates with stable linkages to release the protein in free form. This study conducted a direct comparison of mineral affinity of BP-protein conjugates linked together with cleavable disulfide and non-cleavable thioether linkages. Bovine serum albumin (BSA) was used as a model protein and the desired conjugates were created with N-succinimidyl-3-(2-pyridyldithio)propionate (disulfide) and succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (thioether) linkers. The disulfide-linked conjugates were cleaved in the presence of a major thiol constituent of serum, cysteine. The imparted mineral affinity, as assessed by hydroxyapatite binding in vitro, was lost upon the cleavage of the disulfide-linked aminoBP. The presence of the serum did not accelerate the cleavage of disulfide-linked conjugates. The aminoBP-BSA conjugates formed with disulfide and thioether linkages were subcutaneously implanted in rats with two different mineral-based matrices to assess protein loss from the matrices. All conjugates exhibited a higher retention in mineral matrices as compared to unmodified BSA. However, no significant differences in in situ pharmacokinetics of the disulfide- and thioether-linked conjugates were observed. We conclude that disulfide-linked BP conjugates were readily cleavable by the amino acid cysteine in vitro, but in vivo cleavage of the disulfide-linked conjugates was not evident when the proteins were implanted adsorbed to mineral-based matrices. BP-protein conjugates with faster-cleaving tethers might be required to significantly influence the release of the BP conjugates from the mineral matrices.

Objective: Disturbed sensory gating has been related to attention deficit and greater distractibility in patients with schizophrenia, and dysfunction of the alpha-7 subunit of the cholinergic nicotinic receptor has been discussed as its biological basis. Alzheimer's disease is characterized by a cholinergic deficit, and postmortem studies have reported alpha-7 receptor loss in patients with Alzheimer's disease. In this study, the authors tested whether sensory gating is disturbed in patients with Alzheimer's disease., Method: Suppression of the P50 event-related potential following the second click of a double-click paradigm, a measure of sensory gating, was assessed in 17 Alzheimer's disease patients and 17 comparison subjects., Results: Alzheimer's disease patients showed less P50 suppression following the second click relative to the comparison subjects., Conclusions: Disturbed sensory gating might result from cholinergic dysfunction and possibly from alpha-7 nicotinic receptor loss in patients with Alzheimer's disease. Prospective studies should investigate the relationship between sensory gating deficit and behavioral disturbances in Alzheimer's disease patients.

Background: An accurate evaluation of warm heart surgery cannot be limited to the assessment of the myocardial effects of warm blood cardioplegia but should also address the effects of systemic normothermia on the inflammatory response to cardiopulmonary bypass. A major component of this response is the endothelial adhesion of neutrophils, because it is linked to the release of cytotoxic compounds. This study was designed (1) to characterize the bypass-induced changes in the expression of neutrophil adhesion molecules (L-selectin and beta 2-integrins) and (2) to assess the influence of bypass temperature on these changes., Methods and Results: Twenty case-matched patients undergoing open-heart procedures were divided into two equal groups according to the core temperature during cardiopulmonary bypass: warm (33.4 +/- 0.3 degrees C) or cold (27.1 +/- 0.4 degrees C, P < .0001 versus warm). Arterial blood samples were collected before, during, and 30 minutes after bypass and processed for the expression of L-selectin and beta 2-integrins (CD11a, CD11b, and CD11c) with flow cytometry. Warm bypass was associated with an early and sustained upregulation of CD11b. In contrast, hypothermia resulted in a strikingly less pronounced CD11b upregulation during bypass. However, CD11b expression sharply increased thereafter so that 30 minutes after bypass, it was no longer significantly different between the two groups. Changes in CD11c expression grossly paralleled those described for CD11b. Neither CD11a nor L-selectin changed significantly from baseline values in either group., Conclusions: Clinical cardiopulmonary bypass is associated with a marked upregulation of the neutrophil CD11b and CD11c integrins. Hypothermia delays but does not prevent the increased expression of these adhesion molecules, which could consequently represent logical targets for interventions designed to blunt the neutrophil-mediated component of bypass-induced inflammatory tissue damage.