Surface modified dendrimers: synthesis and characterization for cancer targeted drug delivery

Dendrimers represents a highly branched three-dimensional structure that provides a high degree of surface functionality and versatility. PAMAM dendrimers are used as well-defined nanocontainers to conjugate, complex or encapsulate therapeutic drugs or imaging moieties. Star-burst [PAMAM] dendrimers represent a superior carrier platform for drug delivery. The present study was aimed at synthesis of a surface modified dendrimer for cancer targeted drug delivery system. For this 4.0 G PAMAM dendrimer was conjugated with Gallic acid [GA] and characterized through UV, IR, 1H NMR and mass spectroscopy. Cytotoxicity study of dendrimer conjugate was carried out against MCF-7 cell line using MTT assay. The study revealed that the conjugate is active against MCF-7 cell line and might act synergistically with anti-cancer drug and gallic acid-dendrimer conjugate might be a promising nano-platform for cancer targeting and cancer diagnosis.     

Co-polymeric hydrophilic nanospheres for drug delivery: release kinetics, and cellular uptake

Nanobiotechnology focuses on the biological effects and applications of nanoparticles that include nano-safety, drug encapsulation and nanotherapeutics. The present study focuses on hydrophilic nanospheres of copolymers N-isopropylacrylamide [NIPAAM] and vinyl pyrrolidone [VP], encapsulating a bioactive derivative of 5-fluorouracil-hexyl-carbamoyl fluorouracil (HCFU). The size of the nanospheres was approximately 58 nm and the surface charge measured was -15.4 mV. Under optimal conditions, the yield was >80%, and the drug loading was 2%. The entrapment efficiency was approximately 75%. Wide-angle X-ray diffraction analysis showed that the entrapped HCFU was present in an amorphous state, which has higher water solubility compared with the crystalline state. Slow drug release from nanospheres was observed in PBS and serum, with approximately 80% released at 37 degrees C after 72 h. The HCFU loaded polymeric nanospheres have been found to be stable in whole blood having negligible RBC toxicity. Cytotoxicity in Mia-Paca 3, pancreatic cancer cell line was done in a 24-72 h assay. Dose dependant cytotoxicity was observed when incubated with various concentrations of HCFU loaded polymeric nanospheres while HCFU per se (<1 mg) showed 90% toxicity within 24 h.     

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.     

Synthesis and characterization of a hemoglobin-ribavirin conjugate for targeted drug delivery

A novel conjugate of human hemoglobin (Hb) and the nucleoside analogue ribavirin (RBV) was synthesized to demonstrate the utility of Hb as a biocompatible drug carrier for improved drug delivery in the treatment of liver disease. RBV is used in combination with interferon for the treatment of hepatitis C, but its side effects can result in dose limitation or discontinuation of treatment. Targeted delivery of RBV may help to prevent or minimize its toxicity. The hemoglobin-ribavirin conjugate (Hb-RBV) was designed to release bioactive drug upon endocytosis by cells and tissues involved in extracellular Hb catabolism and clearance. Ribavirin-5'-monophosphate (RBV-P) was prepared from RBV and activated as the 5'-monophosphorimidazolide (RBV-P-Im) for reaction with carbonmonoxyhemoglobin to yield Hb-RBV consisting of multiple RBV drugs covalently attached as physiologically labile phosphoramidates via their 5'-hydroxyl groups. A molar drug ratio of six to eight RBV molecules per Hb tetramer was obtained with near complete haptoglobin (Hp) binding of the drug modified Hb maintained. The conjugate complex (Hp-Hb-RBV) was selectively taken up in vitro by cells that express the hemoglobin-haptoglobin receptor, CD163. Recovered ribavirin enzymatically cleaved from Hb-RBV showed equipotent antiproliferative activity compared to control unconjugated RBV against human HepG2 and mouse AML12 liver cell lines. Based upon the reported high level of Hb uptake in the liver, Hb-RBV may be useful in the treatment of certain liver diseases, as well as inflammatory disorders associated with CD163-positive macrophages.     

Polyester dendritic systems for drug delivery applications: design,synthesis, and characterization

Attachment of drugs to high molecular weight polymers can significantly improve both tumor targeting and therapeutic efficacy due to the enhanced permeability and retention effect observed in tumor tissue. However, the commercial availability of well-defined water-soluble polymeric systems with narrow polydispersities that are biocompatible, nontoxic, and nonimmunogenic is rather limited. To address this need, we have investigated dendritic polymers as promising scaffolds for the preparation of new soluble polymeric drug carriers due to their well-defined molecular architecture and their multiplicity of surface sites. Herein we show the design and synthesis of dendritic polyester systems based on the monomer unit 2,2-bis(hydroxymethyl)propanoic acid as a possible versatile drug carrier. The potent anticancer drug doxorubicin was attached via a pH-sensitive linkage to one of the carriers presented, demonstrating the feasibility of using these polyester dendritic structures to prepare a viable polymer-drug conjugate.     

Nanoparticle-based biocompatible and targeted drug delivery: characterization and in vitro studies

Paclitaxel nanoparticles (PAX NPs) prepared with the size of 110 ± 10 nm and ζ potential of -40 ± 3 mV were encapsulated in synthetic/biomacromolecule shell chitosan, dextran-sulfate using a layer-by-layer self-assembly technique. Zeta potential measurements, analysis of X-ray photoelectron spectroscopy, and scanning electron microscopy confirmed the successful adsorption of each layer. Surface modifications of these core-shell NPs were performed by covalently conjugating with poly(ethylene glycol) (H(2)N-PEG-carboxymethyl, M(w) 3400) and fluorescence labeled wheat germ agglutinin (F-WGA) to build a biocompatible and targeted drug delivery system. 32% of PAX was released from four bilayers of biomacromolecule assembled NPs within 8 h as compared with >85% of the drug released from the bare NPs. Moreover, high cell viability with PEG conjugation and high binding capacity of WGA-modified NPs with Caco-2 cells were observed. This biocompatible and targeted NP-based drug delivery system, therefore, may be considered as a potential candidate for the treatment of colonic cancer and other diseases.     

A new method for targeted drug delivery using polymeric microcapsules

Recent research and clinical evidence suggest that thalidomide could potentially be used to treat inflammation associated with Crohn's disease. However, systemic side effects associated with large doses of this drug have limited its widespread use. Treatment, with thalidomide would prove more efficacious if the drug could be delivered directly to target areas in the gut, thereby reducing systemic circulation. Microcapsule encapsulation could enable direct delivery of the drug. To assess the latter, we designed and tested drug-targeting release characteristics of alginate-poly-l-lysine-alginate (APA) microcapsules in simulated gastrointestinal environments. The results show that APA capsules enabled delivery of thalidomide in the middle and distal portions of the small intestine. We also compared the APA membrane formulation with an earlier designed alginate chitosan (AC) membrane thalidomide formulation. The results show that both APA and AC capsules allow for successful delivery of thalidomide in the gut and could prove beneficial in the treatment of Crohn's disease. However, further research is required.     

Biophysical characterization of a riboflavin-conjugated dendrimer platform for targeted drug delivery

The present study describes the biophysical characterization of generation-five poly(amidoamine) (PAMAM) dendrimers conjugated with riboflavin (RF) as a cancer-targeting platform. Two new series of dendrimers were designed, each presenting the riboflavin ligand attached at a different site (isoalloxazine at N-3 and d-ribose at N-10) and at varying ligand valency. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the binding activity for riboflavin binding protein (RfBP) in a cell-free solution. The ITC data shows dendrimer conjugates have K(D) values of ≥ 465 nM on a riboflavin basis, an affinity ~93-fold lower than that of free riboflavin. The N-3 series showed greater binding affinity in comparison with the N-10 series. Notably, the affinity is inversely correlated with ligand valency. These findings are also corroborated by DSC, where greater protein-conjugate stability is achieved with the N-3 series and at lower ligand valency.     

Solid-phase synthesis, bioconjugation, and toxicology of novel cationic oligopeptoids for cellular drug delivery

For many therapeutic applications, it has become more and more important to find synthetic compounds that have the ability to transport a variety of drugs and cargo molecules into cells and tissues. Like arginine-rich cell-penetrating peptides (CPPs), it is already known that peptide mimetics such as beta-peptides and peptoids can also express a transport function. In this study, ten fluorophore-labeled chiral and achiral peptoids with different backbone lengths and side chains as well as three peptoids coupled to a therapeutically active porphyrin moiety were prepared using a highly modular solid-phase synthesis (SPP) approach. To compare the structural determinants with the cellular uptake efficiency, all peptoids were analyzed by live cell imaging. All cells show an even vesicular distribution of the internalized peptoids, also revealing that a vesicular escape into the cytosol was stronger for peptoids with longer backbones. Moreover, the uptake efficiency correlated with both the incubation time and the given concentration. Toxicology tests and uptake experiments with porphyrin-coupled peptoids indicate their suitability for application as robust and readily available drug delivery systems or intracellular probes.     

Molecular vehicles for targeted drug delivery

Targeted drug delivery by cell-specific cytokines and antibodies promises greater drug efficacy and reduced side effects. We describe a novel strategy for assembly of drug delivery vehicles that does not require chemical modification of targeting proteins. The strategy relies on a noncovalent binding of standardized "payload" modules to targeting proteins expressed with a "docking" tag. The payload modules are constructed by linking drug carriers to an adapter protein capable of binding to a docking tag. Using fragments of bovine ribonuclease A as an adapter protein and a docking tag, we have constructed vascular endothelial growth factor (VEGF) based vehicles for gene delivery and for liposome delivery. Assembled vehicles displayed remarkable selectivity in drug delivery to cells overexpressing VEGF receptors. We expect that our strategy can be employed for targeted delivery of many therapeutic or imaging agents by different recombinant targeting proteins.     

Synthesis of RGD analogs as potential vectors for targeted drug delivery

RGD analogs bind to integrin receptors with high affinity and therefore have the potential to be used as vectors for the targeted delivery of pharmaceutical agents to designated sites. Critical to this application is the ability to synthesize RGD analogs with different side chain functional groups that allow for the ready tethering of pharmaceutical agents without sacrificing their affinity for the target receptor significantly. A series of RGD analogs intended to be used as delivery vectors of pharmaceutical agents were prepared and evaluated for their ability to inhibit platelet aggregation by binding to glycoprotein IIb/IIIa. Among them, compound 11 showed the lowest IC50 against platelets activated by ADP. It was found that such RGD analogs could tolerate side chain modification fairly well with various functional groups attached such as amide, amine, ester, protected amine and poly(ethylene glycol). The fact that the compound with a side chain modification of poly(ethylene glycol) retained high affinity for glycoprotein IIb/IIIa (IC50 150 nM) suggests the feasibility of tethering fairly large pharmaceutical agents to such RGD analogs without significant sacrifice of their affinity to the intended receptor.     

Use of intracellular transport processes for targeted drug delivery into a specified cellular compartment

Targeted drug delivery into the cell compartment that is the most vulnerable to effects of the corresponding drug is a challenging problem, and its successful solution can significantly increase the efficiency and reduce side effects of the delivered therapeutic agents. To accomplish this one can utilize natural mechanisms of cellular specific uptake of macromolecules by receptor-mediated endocytosis and intracellular transport between cellular compartments. A transporting construction combining the components responsible for different steps of intracellular transport is promising for creating multifunctional modular constructions capable of delivering the necessary therapeutic agent into a given compartment of type-specified cells. This review focuses on intracellular transport peculiarities along with approaches for designing such transporting constructions for new, more effective, and safer strategies for treatment of various diseases.     

Silk-fibroin-coated liposomes for long-term and targeted drug delivery

Many barriers to drug delivery into a tumor site require careful consideration when designing a new drug. In this study, the adhesive targeting and drug specificity of modified liposomal vesicles on human-scar-producing cells, keloid fibroblasts, were investigated. Keloids express abundant levels of mucopolysaccharides and receptor tyrosine kinase (RTK). In this report, the structural properties, drug release kinetics, and therapeutic availability of silk-fibroin-coated, emodin-loaded liposomes (SF-ELP), compared with uncoated, emodin-loaded liposomes (ELP), were investigated. SF-ELP had a highly organized lamellae structure, which contributed to 55% of the liposomal diameter. This modified liposomal structure decreased emodin release rates by changing the release kinetics from a swelling and diffusional process to a purely diffusional process, probably due to steric hindrance. SF-ELP also increased adhesion targeting to keloid fibroblasts. Increased retention of SF-ELP is most likely due to the interaction of the fibrous protein coating around the ELP with the pericellular molecules around the cell. SF-ELP also decreased survival rate of keloids that expressed high levels of RTK. These results demonstrated that SF-ELP enhanced emodin delivery by improved diffusion kinetics and specific cell targeting.     

Gold nanoparticles: Emerging paradigm for targeted drug delivery system

The application of nanotechnology in medicine, known as nanomedicine, has introduced a plethora of nanoparticles of variable chemistry and design considerations for cancer diagnosis and treatment. One of the most important field is the design and development of pharmaceutical drugs, based on targeted drug delivery system (TDDS). Being inspired by physio-chemical properties of nanoparticles, TDDS are designed to safely reach their targets and specifically release their cargo at the site of disease for enhanced therapeutic effects, thereby increasing the drug tissue bioavailability. Nanoparticles have the advantage of targeting cancer by simply being accumulated and entrapped in cancer cells. However, even after rapid growth of nanotechnology in nanomedicine, designing an effective targeted drug delivery system is still a challenging task. In this review, we reveal the recent advances in drug delivery approach with a particular focus on gold nanoparticles. We seek to expound on how these nanomaterials communicate in the complex environment to reach the target site, and how to design the effective TDDS for complex environments and simultaneously monitor the toxicity on the basis of designing such delivery complexes. Hence, this review will shed light on the research, opportunities and challenges for engineering nanomaterials with cancer biology and medicine to develop effective TDDS for treatment of cancer.     

Microparticulate systems for targeted drug delivery to phagocytes

Comment on: Jhunjhunwala S, et al. J Control Release 2009;133:191-7.     

Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery

Assembled modular complexes for targeted drug delivery can be based on strong non-covalent interactions between a cargo module containing an adapter protein and a docking tag fused to a targeting protein. We have recently constructed a completely humanized adapter/docking tag system based on interactions between 15 amino acid (Hu-tag) and 110 amino acid (HuS) fragments of human ribonuclease I (RNase I). Although recombinant HuS can be expressed and refolded into a functionally active form, the purification procedure is cumbersome and expensive, and more importantly, it yields a significant proportion of improperly folded proteins. Here we describe engineering, high-yield expression, and purification of a chimeric bovine/human RNase (BH-RNase) comprising 1-29 N-terminal amino acids of bovine ribonuclease A and 30-127 amino acids of human RNase I. Unlike RNase I, the chimeric BH-RNase can be cleaved by either subtilisin or proteinase K between A20 and S21, providing a functionally active HuS. The HuS obtained from chimeric BH-RNase differs from wild-type HuS by an N24T substitution; therefore, we have reverted this substitution by mutating N24 to T24 in BH-RNase. This BH-RNase mutant can also be cleaved by subtilisin or proteinase K yielding wild-type HuS. The affinity of HuS obtained from BH-RNase to Hu-tag is approximately five times higher than that for recombinant HuS, reflecting a higher percentage of properly folded proteins.     

Cancer hallmarks and malignancy features: Gateway for improved targeted drug delivery

Cancer chemotherapy is mainly based on the use of cytotoxic compounds that often affect other tissues, generating serious side effects which deteriorate the quality of life of patients. Recent advancements in targeted drug delivery systems offer opportunities to improve the efficiency of chemotherapy, by the use of smaller drug doses with reduced side effects. In the gene therapy approach, this consists in improving the transformation potential of the gene delivery system. Interestingly, these systems further provide good platforms for the delivery of hydrophobic and low-bioavailability compounds, while facilitating the penetration of the blood-brain barrier. The present report provides an overview of biologically relevant cancer hallmarks that can be exploited to design effective delivery vehicles that release cytotoxic compounds specifically in cancer tissues, in a targeted manner. The relevance of each cancer marker is presented, with particular emphasis on the generation of these hallmarks and their importance in cancer cell biology.     

A photochemical approach for controlled drug release in targeted drug delivery

Photochemistry provides a unique mechanism that enables the active control of drug release in cancer-targeting drug delivery. This study investigates the light-mediated release of methotrexate, an anticancer drug, using a photocleavable linker strategy based on o-nitrobenzyl protection. We evaluated two types of the o-nitrobenzyl-linked methotrexate for the drug release study and further extended the study to a fifth-generation poly(amidoamine) dendrimer carrier covalently conjugated with methotrexate via the o-nitrobenzyl linker. We performed the drug release studies by using a combination of three standard analytical methods that include UV/vis spectrometry, (1)H NMR spectroscopy, and anal. HPLC. This article reports that methotrexate is released by the photochemical mechanism in an actively controlled manner. The rate of the drug release varies in response to multiple control parameters, including linker design, light wavelength, exposure time, and the pH of the medium where the drug release occurs.