Pharmaceutical NanotechnologyDelivery of paclitaxel across cellular barriers using a dendrimer-based nanocarrier
Graphical abstract
Introduction
Over the years, numerous attempts have been made to devise therapeutic drug carrier systems able to cross cellular barriers (e.g. intestinal barrier and the blood–brain barrier) for efficient drug delivery. Challenges encountered during drug delivery are normally associated with low solubility and permeability of therapeutic drugs. Efflux transporter systems (e.g. P-gp efflux transporter) actively function at cellular barriers and limit drugs that are substrates from permeation across the barrier. Chemical modification is one of the strategies to enhance permeability and solubility of drugs for more efficient delivery. Addition of lipophilic components to drugs and conjugation of drug molecules to a more soluble carrier which can bypass efflux transporters have been proven to enhance the permeation of drugs across cellular barriers (Abbott and Romero, 1996, Najlah and D’Emanuele, 2006).
In this study, paclitaxel was selected as a P-gp substrate with low water solubility (<1 μM). It represents a class of anti-microtubule anticancer drugs which have been shown experimentally to have antitumor activity (Rowinsky et al., 1990) by promoting microtubule polymerization, a process which disrupts the normal tubule dynamics essential in cellular division, and leads to cell death by apoptosis (Guillemard and Saragovi, 2001). Paclitaxel has been reported to demonstrate remarkable efficacy against ovarian and breast cancer and more recently, against malignant gliomas and brain metastases (Fellner et al., 2002). Despite its clinical efficacy, pharmaceutical applications of paclitaxel are limited by its poor solubility as well as low permeability due to exclusion by the P-gp efflux transport system present in cellular barriers, e.g. the intestinal and the blood–brain barriers.
Polyamidoamine (PAMAM) dendrimers have shown great potential as drug carriers in pharmaceutical applications due to their well-defined architecture (D’Emanuele et al., 2006, Tomalia et al., 1985). They are highly branched polymers with a high degree of uniformity and monodispersity, and amendable surface groups for specific functionality (D’Emanuele and Attwood, 2005, Tomalia et al., 1990). Their unique properties and characteristics have been of great interest for encapsulation/solubilization of drugs, and conjugation of drugs for transepithelial transport (D’Emanuele et al., 2004). Dendrimers have been reported to cross cellular barriers via both paracellular and transcellular pathways (El-Sayed et al., 2002, El-Sayed et al., 2003, Jevprasesphant et al., 2003a, Kitchens et al., 2005, Kitchens et al., 2007, Saovapakhiran et al., 2009). Goldberg et al. (2010) reported that the internalization of G3.5 PAMAM dendrimers via endocytosis promoted transient tight junction opening, indicating that transcellular and paracellular pathways are interconnected for dendrimer cellular transport. For targeting to tumors, conjugation of paclitaxel to G4-OH PAMAM dendrimers resulted in a 10-fold increase in cytotoxicity in A2780 human ovarian carcinoma cells compared to free drug (Khandare et al., 2006). Partially acetylated G5 PAMAM dendrimer conjugated with paclitaxel, folic acid and FITC demonstrated significant cytotoxicity against KB cells (human epidermoid carcinoma cells that over-express the folate receptor) in cellular uptake and specific delivery studies (Majoros et al., 2006). Previous work in our research group has shown the ability of PAMAM dendrimer conjugates to enhance drug solubility and bypass P-glycoprotein (P-gp) efflux transporters, therefore increasing drug bioavailability (D’Emanuele et al., 2004, Najlah et al., 2007a, Najlah et al., 2007b). G3 PAMAM dendrimer was reported as a potential drug carrier for propranolol, a P-gp substrate drug with low water solubility. Enhanced permeability and ability to bypass the P-gp efflux transporter through Caco-2 cell monolayers were observed when propranolol was conjugated to surface-modified G3 PAMAM dendrimer (D’Emanuele et al., 2004). Surface-engineered PAMAM dendrimers with lauryl chains demonstrated higher permeability and lower cytotoxicity compared to unmodified dendrimers (Jevprasesphant et al., 2003a, Jevprasesphant et al., 2003b). PAMAM dendrimers conjugated to drugs via biodegradable linkers were assessed by Najlah et al., 2006, Najlah et al., 2007a, Najlah et al., 2007b. Diethylene glycol (deg) and succinic acid (suc) were used as the linkers to attach drugs to PAMAM dendrimers. Enhanced solubility and permeability were obtained when naproxen was conjugated to G0 PAMAM dendrimer via a deg linker (Najlah et al., 2007b). Further studies were conducted with the conjugates of terfenadine (a water-insoluble P-gp substrate drug) with lauryl-modified G1 PAMAM dendrimers via a double linker (suc-deg) (Najlah et al., 2007a). The dendrimer prodrugs demonstrated enhanced permeability and solubility, and ability to bypass the P-gp efflux transport system.
There are relatively few studies of permeation of PAMAM dendrimer across the blood–brain barrier (BBB). PEGylated G5 PAMAM dendrimer conjugated with brain-targeting ligands transferrin (Tf) and lactoferrin (Lf), demonstrated increased brain uptake, transfection efficacy and brain gene expression in gene delivery studies. These findings offer a promising non-viral approach for gene delivery to the brain via non-invasive administration (Huang et al., 2007, Huang et al., 2008).
In the present study, dendrimer-based drug delivery systems consisting of lauryl-modified G3 PAMAM dendrimer conjugated with paclitaxel were synthesized and characterized. The synthesis of paclitaxel–dendrimer prodrugs has been reported in which a double ester linkage was employed to attach the drug to dendrimer surface using succinic anhydride/acid linkers (Khandare et al., 2006, Majoros et al., 2006). It has been previously shown that the chemical and enzymatic stability of dendrimer conjugates is related to the type of the linkage (Najlah et al., 2006). The primary ester bond was found to be more labile than the amide bond under the influence of pH and in the presence of esterases. In this study, a glutaric anhydride linker was selected to attach to paclitaxel via an ester bond and then conjugated with PAMAM dendrimer via an amide bond. This ensures that the dendrimer conjugates are stable during transit yet able to release the drug once delivered to the target. The transport and cytotoxicity of dendrimer carriers were investigated using Caco-2 cells and the cytotoxicity and permeability of the dendrimer prodrugs were assessed. Furthermore, the ability of the paclitaxel–dendrimer prodrugs to overcome the blood–brain barrier was investigated using a model based on primary porcine brain endothelial cells (PBECs) (Abbott et al., 2008, Abbott et al., 2010, Patabendige et al., 2012, Skinner et al., 2009).
Section snippets
Materials
Third-generation PAMAM dendrimer (G3) with an ethylene diamine core in methanol (20%, w/w) was purchased from Dendritech Inc. (Michigan, USA). Paclitaxel was purchased from Advance Tech. & Ind. Co., Ltd. (Kln, Hong Kong). Silica gel for flash chromatography was purchased from BDH Laboratory Supplies (Lutterworth, UK). Diphenyl phosphoryl chloride (DPC), N-hydroxysuccinimide (NHS), fluorescein isothiocyanate (FITC) 98%, Sephadex LH-20, fibronectin, Dulbecco's Modified Eagle's Medium (DMEM) low
Synthesis and characterization of G3 PAMAM dendrimer with lauryl and paclitaxel
G3Lx. Lauryl chains were covalently attached to the surface primary amine groups of G3 PAMAM dendrimer via carbamate bonds. The 1H NMR studies showed that lauryl alcohol was conjugated to G3 dendrimer at the appropriate molar ratios (3:1 and 6:1) to yield lauryl-G3 PAMAM dendrimers (G3L3 and G3L6). Lauryl alcohol demonstrated higher stability and provided more consistent yield (Najlah et al., 2006) compared to lauroyl chloride used in the method described by Jevprasesphant et al. (2003a). The
Conclusions
Novel dendrimer-based drug delivery systems consisting of G3 PAMAM dendrimer, a permeability enhancer (lauryl chains), a linker (glutaric anhydride) and paclitaxel were successfully synthesized and characterized. The dendrimer conjugates were demonstrated to have good stability at a range of pHs (1.2, 7.4, and 8.5) after 48 h of incubation. The ester bond linkage of the conjugates was stable under physiological conditions even after 10 days of incubation. Cytotoxicity studies showed that free G3
Acknowledgments
The authors would like to thank Dr. Jeff Penny for providing the Caco-2 cells. We also thank Dr. Adjanie Patabendige for training in the PBEC model. This work was financially supported by the University of Central Lancashire under a science research program.
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