Biotin-Decorated PAMAM G4.5 Dendrimer Nanoparticles to Enhance the Delivery, Anti-Proliferative, and Apoptotic Effects of Chemotherapeutic Drug in Cancer Cells.
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ABSTRACT: Biotin receptors are overexpressed by various types of solid cancer cells and play a significant role in tumor metabolism, growth, and metastasis. Thus, targeting the biotin receptors on tumor cells may enhance the efficiency and reduce the side-effects of chemotherapy. The aim of this study was to develop a biotin-coupled poly(amido)amine (PAMAM) (PG4.5) dendrimer nanoparticle to enhance the tumor-specific delivery and intracellular uptake of anticancer drugs via receptor-mediated endocytosis. We modified PG4.5 with diethylenetriamine (DETA) followed by biotin via an amide bond and characterized the resulting PG4.5-DETA-biotin nanoparticles by 1H NMR, FTIR, and Raman spectroscopy. Loading and releasing of gemcitabine (GEM) from PG4.5-DETA-biotin were evaluated by UV-Visible spectrophotometry. Cell viability and cellular uptake were examined by MTT assay and flow cytometry to assess the biocompatibility, cellular internalization efficiency and antiproliferative activity of PG4.5-DETA-biotin/GEM. Gemcitabine-loaded PG4.5-DETA-biotin nanoparticles were spherical with a particle size of 81.6 ± 6.08 nm and zeta potential of 0.47 ± 1.25 mV. Maximum drug-loading content and encapsulation efficiency were 10.84 ± 0.16% and 47.01 ± 0.71%, respectively. Nearly 60.54 ± 1.99% and 73.96 ± 1.14% of gemcitabine was released from PG4.5-DETA-biotin/GEM nanoparticles after 48 h at the acidic pH values of 6.5 and 5, respectively. Flow cytometry and fluorescence microscopy of cellular uptake results revealed PG4.5-DETA-biotin/GEM nanoparticles selectively targeted cancer cells in vitro. Cytotoxicity assays demonstrated gemcitabine-loaded PG4.5-DETA-biotin significantly reduced cell viability and induced apoptosis in HeLa cells. Thus, biotin-coupled PG4.5-DETA nanocarrier could provide an effective, targeted drug delivery system and selectively convey gemcitabine into tumor cells.
Project description:The natural xanthone α-mangostin (αM) exhibits a wide range of pharmacological activities, including antineoplastic and anti-nematode properties, but low water solubility and poor selectivity of the drug prevent its potential clinical use. Therefore, the targeted third-generation poly(amidoamine) dendrimer (PAMAM G3) delivery system was proposed, based on hyperbranched polymer showing good solubility, high biocompatibility and low immunogenicity. A multifunctional nanocarrier was prepared by attaching αM to the surface amine groups of dendrimer via amide bond in the ratio 5 (G32B12gh5M) or 17 (G32B10gh17M) residues per one dendrimer molecule. Twelve or ten remaining amine groups were modified by conjugation with D-glucoheptono-1,4-lactone (gh) to block the amine groups, and two biotin (B) residues as targeting moieties. The biological activity of the obtained conjugates was studied in vitro on glioma U-118 MG and squamous cell carcinoma SCC-15 cancer cells compared to normal fibroblasts (BJ), and in vivo on a model organism Caenorhabditis elegans. Dendrimer vehicle G32B12gh at concentrations up to 20 µM showed no anti-proliferative effect against tested cell lines, with a feeble cytotoxicity of the highest concentration seen only with SCC-15 cells. The attachment of αM to the vehicle significantly increased cytotoxic effect of the drug, even by 4- and 25-fold for G32B12gh5M and G32B10gh17M, respectively. A stronger inhibition of cells viability and influence on other metabolic parameters (proliferation, adhesion, ATP level and Caspase-3/7 activity) was observed for G32B10gh17M than for G32B12gh5M. Both bioconjugates were internalized efficiently into the cells. Similarly, the attachment of αM to the dendrimer vehicle increased its toxicity for C. elegans. Thus, the proposed α-mangostin delivery system allowed the drug to be more effective in the dendrimer-bound as compared to free state against both cultured the cancer cells and model organism, suggesting that this treatment is promising for anticancer as well as anti-nematode chemotherapy.
Project description:The synthesis, isolation, and characterization of generation 3 poly(amidoamine) (G3 PAMAM) dendrimer containing precise ratios of 5-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dye (n = 1-3) per polymer particle are reported. Stochastic conjugation of TAMRA dye to the dendrimer was followed by separation into precise dye-polymer ratios using rp-HPLC. The isolated materials were characterized by rp-UPLC, MALDI-TOF-MS, and 1H NMR spectroscopy, UV-vis, and fluorescence spectroscopies.
Project description:Human norovirus is a common cause of acute gastroenteritis worldwide, and oysters have been found to be the main carriers for its spread. The lack of efficient pre-treatment methods has been a major bottleneck limiting the detection of viruses in oysters. In this study, we established a novel immunomagnetic enrichment method using polyamidoamine (PAMAM) dendrimer/SA-biotin-mediated cascade amplification for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) detection. We compared the capture efficiency of traditional immunomagnetic enrichment, biotin-amplified immunomagnetic enrichment, and PAMAM dendrimer/SA-biotin-mediated cascade-amplification immunomagnetic enrichment. The optimal capture efficiency of the novel method was 44.26 ± 1.45%, which increased by 183.17% (P < 0.01) and 18.09% (P < 0.05) compared with the first two methods, respectively. Three methods were all applied in detecting norovirus in 44 retail oysters, the detection rate of the PAMAM dendrimer/SA-biotin-mediated method was 25.0%, which was higher than those of traditional IME (15.90%) and SA-biotin-amplified IME (18.80%) by 9.1 and 6.2%, respectively. In conclusion, the novel method can be applied for the rapid detection of norovirus in oysters, which can help reduce the cost and time of detection and improve detection rates.
Project description:Poly(amidoamine) (PAMAM) dendrimer are branched polymers with low degrees of heterogeneity. Current synthesis methods, however, result in substantial batch variability. We present our optimized procedure for post-synthesis (and post-market) purification of a generation 5 PAMAM dendrimer by membrane dialysis and demonstrate its effectiveness and limitations using a representative lot of biomedical grade dendrimer. This method successfully removes trailing generation defect structures, thereby reducing the heterogeneity of the material (PDI reduced from 1.04 to 1.02). Optimized analytical techniques to characterize the unpurified and purified dendrimer are also detailed. The efficiency of the purification method is successfully monitored by these analytics and dendrimer parameters that are critical for subsequent modification reactions and biological evaluation (M(n), M(w), PDI, average number of end groups) obtained. To provide better definition of the variability that should be expected between lots of synthesized material, HPLC traces for three additional commercial lots of dendrimer are also presented.
Project description:A unique asymmetric bow-tie poly(amidoamine) (PAMAM) dendrimer (ABTD) scaffold was designed and developed as a well-defined macromolecular carrier for tumor-targeted drug delivery. The ABTD scaffold in this study consists of a G3-half-dendron (G3-HD) unit and a G1-half-dendron (G1-HD) unit, bearing thiol moiety in each unit and a bis(maleimide) linker unit, which undergo sequential thiol-maleimide coupling to assemble the scaffold. This assembly methodology is applicable to all other combinations of different generations of PAMAM dendrimers. In the prototype ABTD in this study, 16 biotin moieties were tethered to the G3-HD unit and 4 payloads (new-generation taxoid) to the G1-HD via a self-immolative linker to form an ABTD-tumor-targeting conjugate (ABTD-TTC-1). Two other ABTD-TTCs were synthesized, wherein the G1-HD unit was tethered to a fluorescence-labeled taxoid or to a fluorescent probe. These three ABTD-TTCs were constructed by using a common key ABTD 6 bearing a terminal acetylene group in the G1-HD unit, which was fully characterized as a single molecule by high-resolution mass spectrometry and NMR despite its high molecular weight (Mw: 12 876). Then, the click reaction was employed to couple ABTD 6 with a small-molecule payload or fluorescence probe unit bearing a terminal azide moiety. ABTD-TTC-3, as a surrogate of ABTD-TTC-2, showed substantially enhanced internalization into two cancer cell lines via receptor-mediated endocytosis, attributed to multibinding effect. ABTD-TTC-1 exhibited a remarkable selectivity to cancer cells (1400-7500 times) compared to human normal cells, which demonstrates the salient feature and bright prospect of the ABTD-based tumor-targeted drug-delivery system.
Project description:The use of tumor-specific therapeutic agents is a promising option for efficient and safe nonviral gene transfer in gene therapy. In this study, we describe the efficacy of polyamidoamine (PAMAM)-based nonviral gene delivery carriers, namely, an ornithine conjugated PAMAM (PAMAM-O) dendrimer in delivering apoptin, a tumor-specific killer gene, into human hepatocellular carcinoma (HepG2 cells) and dermal fibroblasts. We analyzed the transfection efficiency by the luciferase assay and assessed cell viability in both cell types. The transfection efficiency of the PAMAM-O dendrimer was found to be higher than that of the PAMAM dendrimer. Moreover, the cytotoxicity of the PAMAM-O dendrimer was very low. We treated both cell types with a polyplex of PAMAM-O dendrimer with apoptin, and analyzed its cellular uptake and localization by confocal microscopy. Cell cycle distribution, tetramethylrhodamine, ethyl ester (TMRE) analysis, and transmission electron microscopy imaging showed that apoptin induced cell death in HepG2 cells. We therefore demonstrated that a PAMAM-O/apoptin polyplex can be used as an effective therapeutic strategy in cancer owing to its effectiveness as a suitable nonviral gene vector for gene therapy.
Project description:Commercial generation-five poly(amidoamine) dendrimer material (G5c) was fractionated into its major structural components. Monomeric G5 (G5m; 21--30 kDa) was isolated to compare its functional properties to the G5c material. Diffusion-ordered nuclear magnetic resonance spectroscopy was employed to measure the self-diffusion coefficients and corresponding hydrodynamic radii of G5m and other G5c components as a function of dendrimer size (i.e., molecular weight) and tertiary structure (i.e., generational or oligomeric nature). It was found that the hydrodynamic radius (R(H)) scales with approximate numbers of atoms in the trailing generations, G5m, and oligomeric material at a rate of R(H)∝N(0.35), in good agreement with previous reports of RH scaling for PAMAM dendrimer with generation. G5c materials can be thought of as a heterogeneous mixture of dendrimers ranging in size from trailing generation two to tetramers of G5, approximately the same in size as a G7 dendrimer, with G5m comprising ∼65% of the material. The radius of hydration for G5m was measured to be 3.1 ± 0.1 nm at pH 7.4. The 10% swelling in response to a drop in pH observed for the G5c material was not observed for isolated G5m; however, the isolated G5--G5 dimers had an increase of 44% in R(H), indicating that the G5c pH response results from the increase in R(H) of the oligomeric fraction upon protonation. Finally, the data allow for an experimental test of the "slip" and "stick" boundary models of the Stokes--Einstein equation for PAMAM dendrimer in water.
Project description:Generation 5 poly(amidoamine) (PAMAM) dendrimers were modified by the addition of cyclic RGD targeting peptides and were evaluated for their ability to associate with siRNA and mediate siRNA delivery to U87 malignant glioma cells. PAMAM-RGD conjugates were able to complex with siRNA to form complexes of approximately 200 nm in size. Modest siRNA delivery was observed in U87 cells using either PAMAM or PAMAM-RGD conjugates. PAMAM-RGD conjugates prevented the adhesion of U87 cells to fibrinogen-coated plates, in a manner that depends on the number of RGD ligands per dendrimer. The delivery of siRNA through three-dimensional multicellular spheroids of U87 cells was enhanced using PAMAM-RGD conjugates compared to the native PAMAM dendrimers, presumably by interfering with integrin-ECM contacts present in a three-dimensional tumor model.
Project description:Skeletal muscle is ideally suited and highly desirable as a target for therapeutic gene delivery because of its abundance, high vascularization, and high levels of protein expression. However, efficient gene delivery to skeletal muscle remains a current challenge. Besides the major obstacle of cell-specific targeting, efficient intracellular trafficking, or the cytosolic transport of DNA to the nucleus, must be demonstrated. To overcome the challenge of cell-specific targeting, herein we develop a generation 5-polyamidoamine dendrimer (G5-PAMAM) functionalized with a skeletal muscle-targeted peptide, ASSLNIA (G5-SMTP). Specifically, to demonstrate the feasibility of our approach, we prepared a complex of our G5-SMTP dendrimer with a plasmid encoding firefly luciferase and investigated its delivery to skeletal muscle cells. Luciferase assays indicated a threefold increase in transfection efficiency of C2C12 murine skeletal muscle cells using G5-SMTP when compared with nontargeting nanocarriers using unmodified G5. To further improve the transfection yield, we employed a cationic dynein light chain 8 protein (DLC8)-binding peptide (DBP) containing an internal sequence known to bind to the DLC8 of the dynein motor protein complex. Complexation of DBP with our targeting nanocarrier, that is, G5-SMTP, and our luciferase plasmid cargo resulted in a functional nanocarrier that showed an additional sixfold increase in transfection efficiency compared with G5-SMTP transfection alone. To our knowledge, this is the first successful use of two different functional nanocarrier components that enable targeted skeletal muscle cell recognition and increased efficiency of intracellular trafficking to synergistically enhance gene delivery to skeletal muscle cells. This strategy of targeting and trafficking can also be universally applied to any cell/tissue type for which a recognition domain exists.
Project description:Poly(amidoamine) (PAMAM) (G3) dendrimer was modified into quaternary ammonium salts using tertiary amines with different chain lengths: dimethyldodecyl amine, dimethylhexyl amine, and dimethylbutyl amine using an efficient synthetic route. The antimicrobial activity of these dendrimer ammonium salts against Staphylococcus and E-coli bacteria was examined using the disc diffusion method. It was found that quaternary ammonium salt prepared with the dimethyldodecyl amine exhibits antimicrobial efficacy against Staphalococus and E.coli bacteria.