Preparation and characterizations of self-assembled PEGylated gelatin nanoparticles

The PEGylated gelatin nanoparticles were prepared by self-assembling method and characterized. The gelatin drug carrier was proposed as a targeting drug delivery system with the hypothesis that the gelatin carrier could be degraded by the matrix metalloprotease (MMP) and release the anticancer drug loaded inside carriers around the cancer site. The gelatin nanoparticles proposed in this study were composed of deoxycholic acid (DOCA), monomethoxy polyethylene glycol (MPEG), and gelatin. The carboxyl groups of DOCA and carboxylated MPEG were coupled with amine group of gelatin by dichlorohexylcarbodiimide (DCC) method. One molecule of gelatin coupled with 205 molecules of MPEG and 275 molecules of DOCA. The synthesized gelatin/DOCA/MPEG conjugates (GDM) were ultrasonicated to produce self-assembled nanoparticles. DOCA acted as the hydrophobic core, thereby aggregating gelatin molecules and hydrophilic MPEG chains located at the surface of the nanoparticles. The concentration of GDM, intensity of sonication, sonication time and temperature, all affected to control the particle size in the ultrasonication. The optimum condition was obtained as 1.0 mg/mL of GDM, 28 W for sonication intensity, 3 min of sonication time, and room temperature. The size distribution of particle was found to be 100–1000 nm in this condition. The particles which had a broad size distribution were filtered by 0.2 μm membrane. The product yield of particles having below 200 nm of size was about 30%. After filtration, an average diameter of GDM nanoparticle was 176 nm (155–200 nm).     

Synthesis of a folate functionalized PEGylated poly(propylene imine) dendrimer as prospective targeted drug delivery system

Based on fourth generation diaminobutane poly(propylene imine) dendrimer, a novel targeted drug nanocarrier was prepared, bearing protective PEG chains and a folate targeting ligand. As a control a PEGylated derivative without folate was also synthesized. The encapsulation and release properties of these PEGylated derivatives were investigated employing etoposide, an anticancer hydrophobic drug. Enhanced solubility of etoposide was achieved inside the dendrimeric scaffold which was subsequently released in a controlled manner. These properties coupled with specificity towards the folate receptor and the low toxicity render folate functionalized PEGylated poly(propylene imine) dendrimer promising candidate for targeted drug delivery.     

A nonradioactive assay for type IV collagen degradation

A sensitive assay for type IV collagen degradation using an avidin-biotin sandwich technique is described. Biotinylated type IV collagen is allowed to bind to an avidin-coated microtiter plate. The solution to be assayed is incubated with the biotinylated collagen bound to the avidin plate. Collagen degraded by the solution is released into the supernatant and transferred to a second plate coated with avidin. By addition of biotinylated horseradish peroxidase to this second plate, the amount of collagen degraded is determined. Our assay requires only 0.5 microgram of type IV collagen per microtiter plate and detects nanogram quantities of bacterial collagenase activity.     

Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase.

The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.     

Folate-functionalized unimolecular micelles based on a degradable amphiphilic dendrimer-like star polymer for cancer cell-targeted drug delivery

A folate-functionalized degradable amphiphilic dendrimer-like star polymer (FA-DLSP) with a well-defined poly(L-lactide) (PLLA) star polymer core and six hydrophilic polyester dendrons based on 2,2-bis(hydroxymethyl) propionic acid was successfully synthesized to be used as a nanoscale carrier for cancer cell-targeted drug delivery. This FA-DLSP hybrid formed unimolecular micelles in the aqueous solution with a mean particle size of ca. 15 nm as determined by dynamic light scattering and transmission electron microscopy. To study the feasibility of FA-DLSP micelles as a potential nanocarrier for targeted drug delivery, we encapsulated a hydrophobic anticancer drug, doxorubicin (DOX), in the hydrophobic core, and the loading content was determined by UV-vis analysis to be 4 wt %. The DOX-loaded FA-DLSP micelles demonstrated a sustained release of DOX due to the hydrophobic interaction between the polymer core and the drug molecules. The hydrolytic degradation in vitro was monitored by weight loss and proton nuclear magnetic resonance spectroscopy to gain insight into the degradation mechanism of the FA-DLSP micelles. It was found that the degradation was pH-dependent and started from the hydrophilic shell gradually to the hydrophobic core. Flow cytometry and confocal microscope studies revealed that the cellular binding of the FA-DLSP hybrid against human KB cells with overexpressed folate-receptors was about twice that of the neat DLSP (without FA). The in vitro cellular cytotoxicity indicated that the FA-DLSP micelles (without DOX) had good biocompatibility with KB cells, whereas DOX-loaded micelles exhibited a similar degree of cytotoxicity against KB cells as that of free DOX. These results clearly showed that the FA-DLSP unimolecular micelles could be a promising nanosize anticancer drug carrier with excellent targeting property.     

Cathepsin B1. A lysosomal enzyme that degrades native collagen

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released (14)C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human alpha(2)-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5-5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an alpha-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.     

Preparation of sodium deoxycholate (DOC) conjugated heparin derivatives for inhibition of angiogenesis and cancer cell growth

We describe new DOC (sodium deoxycholate)-heparin nanoparticles for in vivo tumor targeting and inhibition of angiogenesis based on chemical conjugation and the enhanced permeability and retention (EPR) effect. Heparin has been used as a potent anticoagulant agent for 70 years, and has recently been found to inhibit the activity of growth factors which stimulate the smooth muscle cells around tumor. From the results, DOC and heparin were conjugated by bonding carboxyl groups of heparin with amine groups of aminated sodium deoxycholate. Larger antitumor effects of the DOC-heparin VI (8.5 mol of DOC coupled with 1.0 mol heparin) were achieved in animal studies, compared to heparin alone. We confirmed that the conjugated heparin retained its ability to inhibit binding with angiogenic factor, showing a significant decrease in endothelial tubular formation. These results provide new insights into the nontoxic anticancer drug carrier as well as the design of multifunctional bioconjugates for targeted drug delivery.     

Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation.

The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.     

Ubiquitin-Like Protein from Human Placental Extract Exhibits Collagenase Activity

An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.     

A novel solid dosage form of rifampicin and isoniazid with improved functionality

The aim of the present investigation was to develop a novel dosage form of rifampicin and isoniazid to minimize degradation of rifampicin in acidic medium and to modulate the release of rifampicin in the stomach and isoniazid in the intestine. Gastroretentive tablets of rifampicin (150 mg) were prepared by the wet granulation method using hydroxypropyl methylcellulose, calcium carbonate, and polyethylene glycol 4000. The granules and tablets of rifampicin were characterized. Hard gelatin capsules (size 4) containing a compacted mass of isoniazid (150 mg) and dicalcium phosphate (75 mg) were enteric coated. Two tablets of rifampicin and 1 capsule (size 4) of isoniazid were put into a hard gelatin capsule (size 00). The in vitro drug release and in vitro drug degradation studies were performed. Rifampicin was released over 4 hours by zero-order kinetics from the novel dosage form. More than 90% of isoniazid was released in alkaline medium in 30 minutes. The results of dissolution studies with the US Pharmacopeia XXIII method revealed that a substantial amount of rifampicin was degraded from the immediate release capsule containing rifampicin and isoniazid powder owing to drug accumulation in the dissolution vessel and also to the presence of isoniazid. The degradation of rifampicin to 3-formyl rifampicin SV (3FRSV) was arrested (3.6%–4.8% degradation of rifampicin at 4 hours) because of the minimization of physical contact between the 2 drugs and controlled release of rifampicin in acidic medium in the modified Rossett-Rice apparatus. This study concludes that the problem of rifampicin degradation can be alleviated to a certain extent by this novel dosage form. Published: August 24, 2007     

Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin.

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.     

Purification of rheumatoid synovial collagenase and its action on soluble and insoluble collagen.

1. The neutral collagenase released into the culture medium by explants of ehrumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G-200, Sephadex QAE A-50 and Sephadex G-100 superfine. 2. The final collagenase preparation had a specific activity against thermally reconstituted collagen fibrils of 312 mug collagen degraded min-1 mg enzyme protein-1, representing more than a 1000-fold increase over that of the active culture medium. 3. Electrophoresis in polyacrylamide disc-gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase. 4. The molecular weight of the enzyme, as determined by dodecylsulphate-polyacrylamide gel electrophoresis, was 33000. 5. Data obtained from sutdies with the ion-exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme. 6. It did not hydrolyse the synthetic collagen peptide Pz-Pro-Leu-Gly-Pro-D-Arg and non-specific protease activity was absent. 7. The collagenase attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). 8. At 37 degrees C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight. 9. As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta-articular tissues although rates of attack were less than with reconstituted fibrils. 10. The data suggests that pure rheumatoid synovial collagenase at 37 degrees C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non-specific proteases. 11. The different susceptibilities of various collagenous substrates to collagenase attack are discussed.     

A Study on Factors Affecting the Degradation of Magnesium and a Magnesium-Yttrium Alloy for Biomedical Applications

Controlling degradation of magnesium or its alloys in physiological saline solutions is essential for their potential applications in clinically viable implants. Rapid degradation of magnesium-based materials reduces the mechanical properties of implants prematurely and severely increases alkalinity of the local environment. Therefore, the objective of this study is to investigate the effects of three interactive factors on magnesium degradation, specifically, the addition of yttrium to form a magnesium-yttrium alloy versus pure magnesium, the metallic versus oxide surfaces, and the presence versus absence of physiological salt ions in the immersion solution. In the immersion solution of phosphate buffered saline (PBS), the magnesium-yttrium alloy with metallic surface degraded the slowest, followed by pure magnesium with metallic or oxide surfaces, and the magnesium-yttrium alloy with oxide surface degraded the fastest. However, in deionized (DI) water, the degradation rate showed a different trend. Specifically, pure magnesium with metallic or oxide surfaces degraded the slowest, followed by the magnesium-yttrium alloy with oxide surface, and the magnesium-yttrium alloy with metallic surface degraded the fastest. Interestingly, only magnesium-yttrium alloy with metallic surface degraded slower in PBS than in DI water, while all the other samples degraded faster in PBS than in DI water. Clearly, the results showed that the alloy composition, presence or absence of surface oxide layer, and presence or absence of physiological salt ions in the immersion solution all influenced the degradation rate and mode. Moreover, these three factors showed statistically significant interactions. This study revealed the complex interrelationships among these factors and their respective contributions to degradation for the first time. The results of this study not only improved our understanding of magnesium degradation in physiological environment, but also presented the key factors to consider in order to satisfy the degradation requirements for next-generation biodegradable implants and devices.     

Development and <Emphasis Type="Italic">In Vitro</Emphasis> Characterization of Galactosylated Low Molecular Weight Chitosan Nanoparticles Bearing Doxorubicin

The aim of the present research was to evaluate the potential of galactosylated low molecular weight chitosan (Gal-LMWC) nanoparticles bearing positively charged anticancer, doxorubicin (DOX) for hepatocyte targeting. The chitosan from crab shell was depolymerized, and the lactobionic acid was coupled with LMWC using carbodiimide chemistry. The depolymerized and galactosylated polymers were characterized. Two types of Gal-LMWC(s) with variable degree of substitution were employed to prepare the nanoparticles using ionotropic gelation with pentasodium tripolyphosphate anions. Factors affecting nanoparticles formation were discussed. The nanoparticles were characterized by transmission electron microscopy and photon correlation spectroscopy and found to be spherical in the size range 106–320 nm. Relatively higher percent DOX entrapment was obtained for Gal-LMWC(s) nanoparticles than for LMWC nanoparticles. A further increase in drug entrapment was found with nanoparticles prepared by Gal-LMWC with higher degree of substitution. A hypothesis which correlates the ionic concentration of DOX in nanoparticles preparation medium and percent DOX entrapment in cationic polymer has been proposed to explain the enhanced DOX entrapment. In-vitro drug release study demonstrated an initial burst release followed by a sustained release. The targeting potential of the prepared nanoparticles was assessed by in vitro cytotoxicity study using the human hepatocellular carcinoma cell line (HepG₂) expressing the ASGP receptors on their surfaces. The enthusiastic results showed the feasibility of Gal-LMWC(s) to entrap the cationic DOX and targeting potential of developed Gal-LMWC(s) nanoparticles to HepG₂ cell line.     

Apolipophorin III is a substrate for protease IV from Pseudomonas aeruginosa

Our results demonstrated that Pseudomonas aeruginosa serine protease IV degraded apolipophorin III from the haemolymph of Galleria mellonella larvae. ApoLp-III protein was degraded in a stepwise manner. Four intermediate forms of 15, 13.3, 11.9 and 9.5 kDa were detected after 30 min digestion while only one of 5.6 kDa was released after 1-h incubation time. N-terminal amino acid sequence analysis of 5.6 kDa peptide revealed that it was released from apoLp-III after cleavage between lysine 70 and 71. ApoLp-III degradation by protease IV was inhibited by 1 mM TLCK but not 1 mM EDTA, additionally demonstrating that digestion was catalysed by a serine protease. Our data also indicated apoLp-III degradation in vivo during P. aeruginosa infection of G. mellonella larvae.     

Macrophage-fibroblast interactions in collagenase production and cartilage degradation.

Rabbit bone-marrow macrophages and fibroblasts were cultured, independently or together, with pieces of 35S-labelled cartilage or at the surface of dried [14C]collagen gels. Each type of cell, cultivated alone, rapidly degraded the proteoglycan of cartilage, but only the fibroblasts degraded collagen. The co-culture of both types of cell had no consistent effect on the rate of proteoglycan degradation, but it stimulated the rate of collagen degradation. In parallel, the accumulation of collagenase in the culture fluid was enhanced but not that of neutral proteinase. Coinditioned media from macrophage cultures added to cultures of fibroblasts had the same effect as the living macrophages in stimulating the production of collagenase. Their action was itself enhanced when the macrophages had been activated by concanavalin A-stimulated spleen-cell factors. These data suggest that fibroblasts may act as effector cells in producing collagenase and degrading collagen in response to soluble factors released by macrophages under the control of lymphocyte factors.     

Fabrication of agar-gelatin hybrid scaffolds using a novel entrapment method for in vitro tissue engineering applications

Scaffolds of agar and gelatin were developed using a novel entrapment method where agar and gelatin molecules mutually entrapped one another forming stable cell adhesive matrices. Glutaraldehyde was used as a crosslinking agent for gelatin. Three types of hybrid matrices were prepared using agar and gelatin in different proportions in the weight ratio of 1:1, 2:1, and 3:1. Surface characterization of dry scaffolds was carried out by scanning electron microscope. Swelling studies were carried out in phosphate buffer saline (PBS) at physiological pH 7.4. The integral stability of the scaffolds was evaluated by estimating the released disintegrated gelatin from them in PBS at pH 7.4. The attachment kinetics of the cells was evaluated by culturing mouse fibroblast cell line NIH 3T3 on films. The cytocompatibility of these matrices was determined by studying growth kinetics of NIH 3T3 cells on them and morphology of cells was observed through optical photographs taken at various days of culture. It was found that the matrices containing agar and gelatin in 2:1 weight ratio exhibited best growth kinetics. The results obtained from these studies have suggested that the above-described method is a cheap and easy way to fabricate agar-gelatin hybrid scaffolds to grow cells which can be used in various in vitro tissue engineering applications like screening of drugs.     

Preparation and characterization of biopolymeric nanoparticles used in drug delivery

Nanotechnology plays an important role in advanced biology and medicine research particularly in the development of potential site-specific delivery systems with lower drug toxicity and greater efficiency. These include microcapsules, liposomes, polymeric microspheres, microemulsions, polymer micelles, hydrogels, solid nanoparticles etc. In the present study, preparation and characterization of biopolymeric gelatin nanoparticles for encapsulating the antimicrobial drug sulfadiazine and its in vivo drug release in phosphate buffer saline (PBS) have been investigated. The nanoparticles prepared by second desolvation process varied in a size range 200 nm and 600 nm with a drug entrapment efficiency of 50% characterized by atomic force microscopy and dynamic light scattering. The drug release from the nanoparticles occurred up to 30% in a controlled manner.     

Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle

Potent sequence selective gene inhibition by siRNA ‘targeted’ therapeutics promises the ultimate level of specificity, but siRNA therapeutics is hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these problems, ligand-targeted, sterically stabilized nanoparticles have been adapted for siRNA. Self-assembling nanoparticles with siRNA were constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG), as a means to target tumor neovasculature expressing integrins and used to deliver siRNA inhibiting vascular endothelial growth factor receptor-2 (VEGF R2) expression and thereby tumor angiogenesis. Cell delivery and activity of PEGylated PEI was found to be siRNA sequence specific and depend on the presence of peptide ligand and could be competed by free peptide. Intravenous administration into tumor-bearing mice gave selective tumor uptake, siRNA sequence-specific inhibition of protein expression within the tumor and inhibition of both tumor angiogenesis and growth rate. The results suggest achievement of two levels of targeting: tumor tissue selective delivery via the nanoparticle ligand and gene pathway selectivity via the siRNA oligonucleotide. This opens the door for better targeted therapeutics with both tissue and gene selectivity, also to improve targeted therapies with less than ideal therapeutic targets.     

Folate and folate-PEG-PAMAM dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice

Ligand-mediated targeting of drugs especially in anticancer drug delivery is an effective approach. Dendrimers, due to unique surface topologies, can be a choice in this context. In the present study, PAMAM (polyamidoamine) dendrimers up to fourth generation were synthesized and characterized through infrared (IR), nuclear magnetic resonance (NMR), electrospray ionization (ESI) mass spectrometric, and transmission electron microscopic (TEM) techniques. Primary amines present on the dendritic surface were conjugated through folic acid and folic acid-PEG (poly(ethylene glycol))-NHS (N-hydroxysuccinimide) conjugates. Tumor in mice was induced through the use of KB cell culture. Prepared dendritic conjugates were evaluated for the anticancer drug delivery potential using 5-FU (5-fluorouracil) in tumor-bearing mice. Approximately 31% of 5-FU was loaded in folate-PEG-dendritic conjugates. Results indicated that folate-PEG-dendrimer conjugate was significantly safe and effective in tumor targeting compared to a non-PEGylated formulation. Tailoring of dendrimers via PEG-folic acid reduced hemolytic toxicity, which led to a sustained drug release pattern as well as highest accumulation in the tumor area.