Current Pharmaceutical Design - Volume 16, Issue 21, 2010

Delivery of therapeutically relevant agents such as anti-cancer drugs, proteins, genes, siRNA, growth factors, etc has evolved tremendously through rational pharmaceutical design starting from drug discovery to delivery strategies. As diverse are the array of agents to be delivered and their applications, so are the formulations designed. The complexities in developing suitable delivery formulations are unforeseen. For instance, targeted drug delivery to cure various pathological conditions asks for highly specific targeting, overcoming barriers such as the blood brain barrier (BBB), tumor cell penetration, high gene transfection efficiency, high circulation time in the blood, etc. Also, tissue engineering to reconstruct damaged or lost organs demands drug/gene delivery capability and scaffolding. These examples are testimonial to the emergence of a plethora of pharmaceutical formulations and also an eye opener to academia's recent contributions in the field. This review series issue aims to introduce a few current pharmaceutical formulations dedicated to targeted and local drug delivery, and tissue engineering applications and their potential implications towards clinical development. Nanoparticulate formulations for drug delivery are the most popular for they have the ability to be taken up by the target due to their size and charge, reduced elimination by the reticuloendothelial system (RES), and easy administration. Yoo and coworkers [1] have reviewed the factors that control the systemic circulation of NPs and recent progress. They infer that a combination of strategies need to be applied to achieve significant improvement in circulation time such as size, surface shielding, mechanical properties, shape and exploitation of cells, etc. Gan and co-authors [2] have developed a system of docetaxel-loaded nanoparticles (NPs) of biodegradable copolymer, poly(lactic acid)-d-α- tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS). The synthesis of the NPs by TPGS-emulsified PLA-TPGS was controllable and resulted in the desired NP size, higher stability and drug encapsulation efficiency in comparison to PVA-emulsified NPs. The NPs also demonstrated sustainable drug release, dose- and time-dependent cellular uptake by the MCF-7 cancer cells suggesting an internalization mechanism of endocytosis. Also, in comparison to Taxotere®, the NPs provided significantly higher therapeutic effect and lower side effect in vivo. Nanoparticulate targeted therapy by conjugating molecular markers differentially expressed on the cancerous cells has been reported by Phongpradist and coauthors [3] to achieve improved efficacy in leukemia therapeutics. They conjugated cIBR, a cyclic peptide specific for leukocyte function associated antigen-1 (LFA-1), to the surface of non-cytotoxic and colloidally stable nanoparticles. cIBR-coupled nanoparticles showed rapid binding and significantly higher uptake by leukemic cells as compared to untargeted nanoparticles. The level of LFA-1 expression level dictated the binding and internalization of targeted nanoparticles. The ability of nanoparticles to cross the bloodbrain- barrier (BBB) and achieve drug penetration into brain tumor is reported by Xie and coauthors [4]. Paclitaxel-loaded PLGA-based nanoparticles of around 200 nm developed by direct dialysis with different additives (vitamin E TPGS and polysorbate 80) were shown to enhance cellular uptake by MDCK monolayer more than surface-coated particles and inhibit C6 glioma cells on the topical side. Development of non-viral gene delivery vectors for clinical applications is on the rise. Recently, Khan and coworkers [5] have developed low molecular weight polymer with branched polyamidoamines with disulfide bonds and demonstrated complete retardation of DNA mobility at N/P of 5. They also demonstrated significantly higher gene transfection efficiency due to higher protonable nitrogen content and insignificant cytotoxicity in comparison to the conventional PEI vectors. On the basis of the cellular uptake and intracellular disposition mechanisms of the genes and carriers, Xu and coworkers [6] have proposed various modification and cell-targeting strategies to improve chitosan and polyamidoamies as dendrimers for gene delivery. They infer that the development of efficient non-viral vector mediated gene delivery depends on a good understanding of the uptake pathways, effects of the gene carrier characteristics on cellular entry and molecular crowding. A versatile protein drug delivery strategy “ATTEMPTS“ was developed by Huang and coauthors [7] by conjugating protein drugs to cell-penetrating peptides (CPPs). The initially disabled conjugates (prodrug) would start releasing the drug only after the complexes accumulate in the tumor sites by the antibody targeting function and was proven in both protein drugs and polymer-drugs with regulatable cell penetrating behavior. In another study, Hassan and Lau [8] have shed light upon the implications of drug/carrier properties, their effects on the particle dispersity and deposition in the lung in a pulmonary drug delivery scenario. Finally, Chen and Tong coworkers [9] have shown that degradable scaffolds which can release biomolecules with high bioactivity, give in for the growth of tissue are well suited for drug delivery in tissue engineering. Such scaffolds could be fabricated by a variety of techniques such as cross-linking, electrospinning, etc. They conclude that the interactions between cells and scaffolds often dictate the efficacy of drug delivery in tissue engineering.

Numerous types of nanoparticles are being designed for systemic and targeted drug delivery. However, keeping nanoparticles in blood for sufficiently long times so as to allow them to reach their therapeutic target is a major challenge. Upon administration into blood, nanoparticles are quickly opsonized and cleared by the macrophages, thereby limiting their circulation times. Surface-modification of nanoparticles by PEG was developed as the first strategy to prolong nanoparticle circulation. While PEGylation has helped prolong particle circulation, it has several limitations including transient nature of the effect and compromised particle-target interactions. Accordingly, several other approaches have been developed to prolong nanoparticle circulation in blood. These include modification with CD47, modulation of mechanical properties, engineering particle morphology and hitchhiking on red blood cells. In this review, we discuss the factors that affect nanoparticle circulation time and discuss recent progress in development of strategies to prolong circulation time.

The clinical dosage formulation of Paclitaxel and Docetaxel, Taxol® and Taxotere®, while having high efficacy, can cause serious side effects due to the adjuvant used. We have developed a system of nanoparticles (NPs) of biodegradable copolymer, poly(lactic acid)-d-α-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS), for Docetaxel formulation to achieve enhanced, sustainable and controlled chemotherapeutic effectiveness and reduce the undesirable side actions. Docetaxel-loaded PLA-TPGS NPs were synthesized with desired size and physicochemical and pharmaceutical properties. In vitro studies using MCF-7 cancer cells have demonstrated the higher cellular uptake and therapeutic effects of the NP formulation. In vivo pharmacokinetics and biodistribution analysis have shown that one dose of the NP formulation of Docetaxel can achieve a 360-h effective chemotherapy with 3.44-fold higher therapeutic effect and 4.42-fold lower side effect than that of Taxotere® at the same dose of 10 mg/kg, as indicated by the larger area-under-the-curve and better biodistribution.

Leukemia therapeutics are aiming for improved efficacy by targeting molecular markers differentially expressed on cancerous cells. Lymphocyte function-associated antigen-1 (LFA-1) expression on various types of leukemia has been well studied. Here, the role and expression of LFA-1 on leukemic cells and the possibility of using this integrin as a target for drug delivery is reviewed. To support this rationale, experimental results were also included where cIBR, a cyclic peptide derived from a binding site of LFA-1, was conjugated to the surface of polymeric nanoparticles and used as a targeting ligand. These studies revealed a correlation of LFA-1 expression level on leukemic cell lines and binding and internalization of cIBR-NPs suggesting a differential binding and internalization of cIBR-NPs to leukemic cells overexpressing LFA-1. Nanoparticles conjugated with a cyclic peptide against an accessible molecular marker of disease hold promise as a selective drug delivery system for leukemia treatment.

Paclitaxel appears to be a potential substrate of the multidrug resistance protein p-glycoprotein, thus preventing itself from entry into the brain and penetrating blood-brain barrier poorly. In this study, the main objective was to design paclitaxel formulation using PLGA-based nanoparticles with different additives and surface coatings to facilitate the paclitaxel transport through MDCK cell monolayer. PLGA-based nanoparticles of around 200 nm without and with additives and surface coatings were developed by direct dialysis. The transendothelial electrical resistance (TEER) variation of MDCK cell monolayer on the cell inserts imposed by paclitaxel-loaded nanoparticles with and without additives was investigated. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) (MTT) assay was used to quantify the cell viability of C6 glioma cells after administration of formulations on the topical side. Investigations showed that particles with additives were able to enhance cellular uptake more than surface-coated particles. TEER values dropped upon the introduction of paclitaxel-loaded PLGA nanoparticles to the cell inserts. After incubation for 24 h, the cell viability of C6 glioma cells in the wells treated with nanoparticles was lower than that of the control wells without particles. Taken together, PLGA nanoparticles with vitamin E TPGS and polysorbate 80 as additives were successfully obtained as paclitaxel formulations, demonstrating great potential for the delivery of paclitaxel through MDCK cell monolayer in vitro.

DNA condensation, endosomal escape of DNA/polymeric complexes, and unpacking of DNA are the key steps in the process of non-viral gene delivery. Amongst these steps, currently the unpacking of the DNA cargo from the DNA/polymeric nanocomplexes is the most challenging and arguably the most crucial if one wants to achieve high gene transfection with minimum cytotoxicity in the target cell. In this report we review current and past examples in the literature that demonstrate concerted efforts in designing and synthesizing various forms of cationic polymeric vectors having “built in” features. Such features can be certain types of chemical functional groups, such as amines and acids or other degradable bonds like esters, carbonates and disulfides, which allow for breakdown of polymeric vectors in certain cellular compartments. This may lead the DNA cargo to dissociate from the DNA/polymer complexes so as to maximize intracellular gene delivery. Furthermore, we provide further evidence that it is possible to achieve the goal of high gene transfection coupled with low cytotoxicity via rational design and formulation of branched polyamidoamines containing disulfide bonds. The DNA binding ability of these polymers and particle size as well as zeta potential of their DNA complexes were investigated. The cytotoxicity of pure polymer and polymer/DNA complexes at various polymer concentrations was studied in HEK293 human embryonic kidney, HepG2 human liver carcinoma, 4T1 mouse breast cancer and HeLa human cervical cancer cell lines. In vitro gene transfection efficiency induced by polymer/DNA complexes was explored in these cell lines by using luciferase and GFP reporter genes in comparison with PEI.

Gene therapy is a potential medical solution that promises new treatments and may hold the cure for many different types of diseases and disorders of the human race. However, gene therapy is still a growing medical field and the technology is still in its infancy. The main challenge for gene therapy is to find safe and effective vectors that are able to deliver genes to the specific cells and get them to express inside the cells. Due to safety concerns, synthetic delivery systems, rather than viral vectors, are preferred for gene delivery and significant efforts have been focused on the development of this field. However, we are faced with problems like low gene transfer efficiency, cytotoxicity and lack of cell-targeting capability for these synthetic delivery systems. Over the years, we have seen a variety of new and effective polymers which have been designed and synthesized specifically for gene delivery. Moreover, various strategies that aimed at enhancing their physicochemical properties, improving transfection efficiency, reducing cytotoxicity as well as incorporating functional groups that offer better targetability and higher cellular uptake are established. Here, we look at two potential polymeric carriers, chitosan and poly(amidoamine) dendrimers, which have been widely reported for gene delivery. For chitosan, the interest arises from their availability, excellent non-cytotoxicity profile, biodegradability and ease of modification. For poly(amidoamine) dendrimers, the interest arises from their ease of synthesis with controlled structure and size, minimal cytotoxicity, biodegradability and high transfection efficiencies. The latest developments on these polymers for gene delivery will be the main focus of this article.

In order to reduce systemic toxicity and effectively deliver macromolecular drug into tumor cells, a system termed “ATTEMPTS” (antibody targeted, [protamine] triggered, electrically modified prodrug-type strategy) was developed in our laboratory. This approach was adapted from our previously reported heparin/protamine-based system for controlled delivery of protease drugs such as tissue- specific plasminogen activator (tPA). In this “ATTEMPTS” system, the cell-permeable protein drugs are synthesized by conjugating proteins to cell-penetrating peptides (CPPs). Cell penetration ability of such CPP-protein conjugates would initially be disabled, acting as a “prodrug”, by forming polyelectrolyte complexes with a functionalized heparin-antibody moiety. The complexes would accumulate in tumor sites by the antibody targeting function, and then the local release of CPP-protein conjugates would be triggered by protamine. We applied this system to the macromolecular anticancer agents, such as the protein drugs (gelonin and asparaginase) as well as the polymerdrugs (polyrotaxane-doxorubicin and polyrotaxane-camptothecin). Both in vitro and preliminary in vivo studies demonstrated the regulable cell penetration behavior based on the competitive ionic interactions between CPP/heparin and heparin/protamine. Thus, this ATTEMPTS approach provides a multi-functionalized system incorporating the features of targeting, prodrug-like, triggerable release, and cell penetration ability for the delivery of macromolecular anticancer agents. A summary of our work on “ATTEMPTS” is presented in this review.

Dry powder inhalation has good potential to be an alternative route of drug administration, especially for the treatment of lung and systemic diseases. However, typically only a small fraction of the dose can be delivered to the lower airways. Continuous effort on the drug formulation is undergoing to improve the deep lung deposition. Key properties of the drug and carrier particles and their relationships with the particle dispersibility and deposition properties in the lung are discussed individually. Some recent drug formulation techniques for dry powder inhalation are also reviewed.

The formulation and fabrication methods for several types of tissue engineering scaffolds with drug delivery capabilities are presented in this review. Tissue engineered constructs are temporary substitutes developed to treat damaged or lost tissue. One key component of such constructs is scaffolds that are often developed to mimic the extra cellular matrix (ECM). As natural ECM contains biomolecules to support proper growth and function of cells, inclusion of these biomolecular cues have been shown to be necessary for proper cell growth and function in tissue engineering. Thus, an effective tissue engineering scaffold should provide such biomolecular cues. This can be achieved through drug delivery in scaffolds. Studies have shown that drug delivery systems are necessary to protect drugs, and provide sustained drug release that is often needed for effective therapy. The tissue engineering features of 4 scaffold types are described, including films, hydrogels, fibers, and microspheres/nanospheres. Fabrication techniques and drug encapsulation methods for these scaffolds are reviewed in addition to some observations arising from the use of these techniques and methods.