Delivery Of Macromolecular Drugs Research Articles

Enhanced delivery of macromolecular antitumor drugs to tumors by nitroglycerin application

Dose regimens of anticancer agents are usually designed on the basis of the maximum tolerable drug doses, and toxicity prevents drug usage at higher doses, even though the drugs may be more effective at the higher doses. We previously studied macromolecular anticancer drugs, i.e. those larger than 40 kDa, and observed their accelerated accumulation in tumors. Their concentration in tumors was more than 5-100-fold their blood concentration because of the enhanced permeability and retention (EPR) effect. Here, we report that the EPR effect was enhanced by applying nitroglycerin (NG) ointment on the skin of tumor-bearing animals. Tumors studied included breast cancer, which was induced in Sprague-Dawley rats by the chemical carcinogen 7,12-dimethylbenz[a]anthracene, and three different transplanted tumor models in mice. NG was applied on tumor or nontumorous normal skin as well. Two to three times more putative macromolecular drug (an Evans blue/albumin complex) was delivered to solid tumors with NG than without NG. We also demonstrated that NG enhanced tumor delivery with another macromolecular drug candidate, PZP, i.e. polyethylene glycol-conjugated zinc protoporphyrin IX, which inhibits heme oxygenase-1. In addition, we investigated the therapeutic effect of NG using a combination with low molecular weight anthracycline or high molecular weight PZP in mouse tumor models. NG had no apparent toxicity at the doses used, and showed significantly increased therapeutic effects in both cases. Regardless of its site of application, NG thus enhanced the delivery of the drug to tumors, and enhanced therapeutic effects.

ScFv Antibody-induced Translocation of Cell-surface Heparan Sulfate Proteoglycan to Endocytic Vesicles

Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (alphaHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [(35)S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.

Macromolecular Drug Delivery. Edited by Mattias Belting.

Macromolecular Drug Delivery. Edited by Mattias Belting.

Characteristics of Biodegradable Implantable Drug Delivery System with Micro-Porous Structure

To eliminate the release-delayed phenomenon of macromolecular drug delivery system at the early stage, biodegradable implantable drug delivery system with micro-porous structure was designed by Bio-MEMS technology in this paper. Water absorption and degradation characters of polylactic acid - polyglycolic acid (PLGA) material with different monomer ratio were studied. Drug release model was built by mass transfer equation and the Finite Element Method. Influencing factors about the cumulative amount of release drug were analyzed. And the results suggest that with more ratio of polylactic acid(PLA), PLGA would have more greater amount of water absorbed as well as a longer degradation time. Better linearity of cumulative amount of release drug would be achieved by reducing the diameter and number of micro-porous.

Thrombin-sensitive photodynamic agents: A novel strategy for selective synovectomy in rheumatoid arthritis

Protease-sensitive macromolecular prodrugs have attracted interest for bio-responsive drug delivery to sites with up-regulated proteolytic activities such as inflammatory or cancerous lesions. Here we report the development of a novel polymeric photosensitizer prodrug (T-PS) to target thrombin, a protease up-regulated in synovial tissues of rheumatoid arthritis (RA) patients, for minimally invasive photodynamic synovectomy. In T-PS, multiple photosensitizer units are tethered to a polymeric backbone via short, thrombin-cleavable peptide linkers. Photoactivity of the prodrug is efficiently impaired due to energy transfer between neighbouring photosensitizer units. T-PS activation by exogenous and endogenous thrombin induced an increase in fluorescence emission by a factor of 16 after in vitro digestion and a selective fluorescence enhancement in arthritic lesions in vivo, in a collagen-induced arthritis mouse model. In vitro studies on primary human synoviocytes showed a phototoxic effect only after enzymatic digestion of the prodrug and light irradiation, thus demonstrating the functionality of T-PS induced PDT. The developed photosensitizer prodrugs combine the passive targeting capacity of macromolecular drug delivery systems with site-selective photosensitizer release and activation. They illuminate lesions with pathologically enhanced proteolytic activity and induce cell death, subsequent to irradiation.

The augmentation of intracellular delivery of peptide therapeutics by artificial protein transduction domains

Protein transduction domains (PTDs), such as HIV-derived Tat, have been successfully used as functional biomaterials for intracellular delivery of anti-cancer macromolecular drugs (protein, peptides, and oligonucleotides). Although there were therefore great expectations regarding the therapeutic potential of PTDs for the development of anti-cancer therapeutics, their clinical application so far has been extremely limited because of the relatively high concentrations required to mediate any effects on cancer cells in vitro or in vivo. In this context, improving the transduction efficiency of PTDs using phage display-based molecular evolution techniques may be useful for creating artificial PTDs with high efficiency and safety. Here, we report an evaluation of transduction efficiency and toxicity of such artificial PTDs (designated mT02 and mT03) compared with Tat. The internalization of mT02 was the most rapid and efficient by a mechanism different from the usual macropinocytosis. Furthermore, we found that artificial PTDs fused with survivin antagonistic peptide potentiate tumor cell-cytostatic activity. Thus, the results of this work provide new insights for designing new-generation peptide therapeutics for a wide variety of cancers as well as those expressing survivin.

The ATTEMPTS delivery systems for macromolecular drugs

Aiming at successful targeted drug delivery – a system that possesses both targeting and prodrug features that can be activated once the system reaches the target site upon systemic administration – would be desired to reduce systemic toxicity. Previously we proposed a heparin/protamine-based system for delivery of protease drugs such as tissue-specific plasminogen activator (t-PA). This approach, termed ‘antibody targeted, triggered, electrically modified prodrug-type strategy’ (ATTEMPTS), would permit antibody-directed administration of inactive t-PA and allow a subsequent triggered release of the active t-PA at the target site. This system can be adapted to target tumor tissues when protein transduction domain (PTD) peptide such as TAT is incorporated in the ATTEMPTS construct. Both in vitro and preliminary in vivo studies using TAT-gelonin (TAT-Gel) and TAT-asparaginase (TAT-ASNase) conjugates have demonstrated that the on/off regulation of the membrane translocation activity of PTD at tumor target, followed by intracellular delivery of cytotoxic macromolecular drug, can be accomplished. Hence, the PTD-mediated delivery system derived from our previous ATTEMPTS approach is a system that incorporates all of the targeting function, prodrug feature, release mechanism and cell entry mechanism and could become a generic system for delivery of macromolecular drugs.

Hyperthermia-Induced Targeting of Thermosensitive Gene Carriers to Tumors

Locoregional hyperthermia (HT) can be used for site-directed activation of macromolecular drug delivery systems. We have developed a gene delivery system based on thermosensitive block copolymers (TSCs) with a phase transition temperature of 42 degrees C [Zintchenko, A., Ogris, M., and Wagner, E. (2006). Bioconjug. Chem. 17, 766-772], in which the statistical copolymer of vinylpyrrolidinone and N-isopropylacryamide is grafted on polyethylenimine (PEI). Here we applied polyplexes consisting of plasmid DNA and TSCs systemically in A/J mice bearing a syngeneic Neuro2A neuroblastoma tumor subcutaneously in each hind limb. One limb was selectively treated by HT at 42 degrees C, at the same time that polyplexes were injected via the tail vein. Hyperthermia led to increased accumulation of thermosensitive polymer and aggregation of thermosensitive polyplexes in HT-treated tumors, resulting in up to 10-fold increased DNA deposition compared with non-HT-treated tumor. The level of transgene expression induced by TSC polyplexes in HT-treated tumors was significantly higher and selective for tumor tissue. With nonthermosensitive PEI polyplexes HT did not influence transgene deposition or expression in tumor.

Targeted Delivery of Macromolecular Drugs: Asialoglycoprotein Receptor (ASGPR) Expression by Selected Hepatoma Cell Lines used in Antiviral Drug Development

The asialoglycoprotein receptor (ASGPR), an endocytotic cell surface receptor expressed by hepatocytes, is triggered by triantennary binding to galactose residues of macromolecules such as asialoorosomucoid (ASOR). The capacity of this receptor to import large molecules across the cellular plasma membrane makes it an enticing target for receptor-mediated drug delivery to hepatocytes and hepatoma cells via ASGPR-mediated endocytosis. This study describes the preparation and characterization of (125)I-ASOR, and its utility in the assessment of ASGPR expression by HepG2, HepAD38 and Huh5-2 human hepatoma cell lines. ASOR was prepared from human orosomucoid, using acid hydrolysis to remove sialic acid residues, then radioiodinated using iodogen. (125)I-ASOR was purified by gel column chromatography and characterized by SDS-PAGE electrophoresis. The ASOR yield by acid hydrolysis was 75%, with approximately 87 % of the sialic acid residues removed. Electrophoresis and gel chromatography demonstrated substantial differences in (125)I-ASOR quality depending on the method of radioiodination. ASGPR densities per cell were estimated at 76,000 (HepG2), 17,000 (HepAD38) and 3,000 (Huh-5-2). (125)I-ASOR binding to ASGPR on HepG2 cells was confirmed through galactose- and EDTA- challenge studies. It is concluded that (125)I-ASOR is a facilely-prepared, stable assay reagent for ASGPR expression if appropriately prepared, and that HepG2 cells, but not HepAD38 or Huh-5-2 cells, are suitable for studies exploiting the endocytotic ASGPR.

Novel Sterecomplexed Sol–Gel Transition Hydrogels Prepared from Physical Packing of Self-Assembled PEO-PPO-PEO and PPO-PEO-PPO Copolymer Nanoscale Micelles

A new class of stereocomplex-induced hydrogels was synthesized based on self-assembled PEO-PPO-PEO and PPO-PEO-PPO copolymers having multiple hydroxyl groups in the PPO segment. The copolymers were grafted with enantiomeric oligo(L-lactic acid) or oligo(D-lactic acid) chains using ring opening polymerization onto the PPO segment. WAXS and DSC analysis were used to investigate the stereocomplex formation of the equimolar mixture of the copolymers. The copolymer hydrogels having stereocomplexed crystalline domains as crosslinking loci exhibited significantly altered temperature-sensitive sol-gel transition behaviors, and showed more delayed erosion and sustained protein release behaviors, compared to Pluronic F127 copolymer hydrogels. The current hydrogels are expected to be used as useful injectable hydrogel materials for macromolecular drug delivery.

Potential prospects of chitosan derivative trimethyl chitosan chloride (TMC) as a polymeric absorption enhancer: synthesis, characterization and applications

In recent years, researchers have been working extensively on various novel properties of polymers to develop increased efficiency of drug delivery and improve bioavailability of various drug molecules, especially macromolecules. Chitosan, a naturally occurring polysaccharide, because of its protonated/polymeric nature, provides effective and safe absorption of peptide and protein drugs. Its transmucosal absorption is, however, limited to acidic media because of its strong intermolecular hydrogen bonds. A new partially quaternized chitosan derivative, N-trimethyl chitosan chloride (TMC), has been synthesized with improved solubility, safety and effectiveness as an absorption enhancer at neutral pH and in aqueous environment. It enhances the absorption, especially of peptide drugs, by reversible opening of tight junctions in between epithelial cells, thereby facilitating the paracellular diffusion of peptide drugs. This derivative thus opens new perspectives as a biomaterial for various pharmaceutical applications/drug delivery systems. This review deals with the potential use of the quaternized chitosan derivative as a permeation enhancer for the mucosal delivery of macromolecular drugs along with its other biomedical applications.

Hyperthermia induced targeting of thermosensitive gene carriers to tumors

Locoregional hyperthermia (HT) can be used for site-directed activation of macromolecular drug delivery systems. We have developed a gene delivery system based on thermosensitive block copolymers (TSCs) with a phase transition temperature of 42 degrees C [Zintchenko, A., Ogris, M., and Wagner, E. (2006). Bioconjug. Chem. 17, 766-772], in which the statistical copolymer of vinylpyrrolidinone and N-isopropylacryamide is grafted on polyethylenimine (PEI). Here we applied polyplexes consisting of plasmid DNA and TSCs systemically in A/J mice bearing a syngeneic Neuro2A neuroblastoma tumor subcutaneously in each hind limb. One limb was selectively treated by HT at 42 degrees C, at the same time that polyplexes were injected via the tail vein. Hyperthermia led to increased accumulation of thermosensitive polymer and aggregation of thermosensitive polyplexes in HT-treated tumors, resulting in up to 10-fold increased DNA deposition compared with non-HT-treated tumor. The level of transgene expression induced by TSC polyplexes in HT-treated tumors was significantly higher and selective for tumor tissue. With nonthermosensitive PEI polyplexes HT did not influence transgene deposition or expression in tumor.

PTD-modified ATTEMPTS system for enhanced asparaginase therapy: A proof-of-concept investigation

Macromolecular drugs such as proteins and gene products are presumably the most desirable therapeutic agents due to their unmatched substrate specificity and reaction efficiency. Yet, clinical use of these drugs has met with limited success, primarily due to the impermeable nature of the cell membrane that restricts cellular drug uptake to only small (< 600 Da) and hydrophobic molecules. The recent discovery of the protein transduction domain (PTD) membrane-penetrating peptides, such as HIV-TAT, has finally offered the possibility of resolving this cell-membrane barrier for macromolecular drug delivery. Via covalent linkages, these PTD peptides have been shown to ferry the attached macromolecular species across membranes of all cell types, both in vitro and in vivo. Nevertheless, the lack of selectivity for PTD-mediated internalization restricts the application of this cell uptake method in clinical practice, due to concerns of inducing systemic toxicity caused by the carried drugs. Presented herein is a modified version of our previously established “ATTEMPTS” approach in delivery of macromolecular drugs, which integrates the cell-penetrating PTDs into a heparin/protamine-regulated delivery system. In vitro findings using asparaginase (ASNase) as a model macromolecular anti-tumor agent were able to validate the feasibility of this delivery system. The chemically constructed TAT-ASNase conjugates not only were able to translocate into the MOLT-4 cells and elicit the cytotoxic effects, but also this PTD-mediated intracellular drug uptake could be regulated (with on/off control) by the addition of heparin and protamine. This modified ATTEMPTS system therefore presents a new avenue of treatment of various types of cancers and other diseases with macromolecular drugs. In vitro characterization and a preliminary proof-of-concept animal investigation that demonstrates the feasibility of this PTD-mediated ASNase therapeutic system is subsequently described.

Dynamics of photoinduced endosomal release of polyplexes

Endosomal escape is a well-known bottleneck for successful delivery of macromolecular drugs and genes. Photochemical disruption of endosomal membranes is an approach to overcome this bottleneck. In this study, we used the photosensitizer disulphonated meso-tetraphenylporphine with sulfonate groups on adjacent phenyl rings (TPPS 2a) to investigate photoinduced endosomal release in living cells with high resolution fluorescence wide-field microscopy in real time. We studied the release dynamics of 10 kDa dextran and polyplexes consisting of DNA condensed with the cationic polymers linear polyethyleneimine (LPEI), poly-( l)-lysine (PLL) or poly-( d)-lysine (PDL). By means of dual-color microscopy and the use of double-labeled polyplexes DNA and polymer were imaged simultaneously. We show that the characteristics of the cationic polymer significantly influence the release behavior of the polyplexes. The release of dextran occurred within 100 ms. For LPEI/DNA particles, LPEI quickly spread throughout the cytosol similar to dextran, whereas DNA was released slowly (within 4 s) and remained immobile thereafter. In case of PLL particles, both DNA and polymer showed quick release. PDL particles remained condensed upon photosensitizer activation. In addition, we demonstrate that TPPS 2a has biological side effects. Besides stop of microtubule dynamics in the dark, the movement of endosomes ceased after photosensitizer activation.

Preclinical modeling of cytosine arabinoside response inMll-Enltranslocator mouse leukemias

Mouse models of human cancer are a potential preclinical setting for drug testing and for development of methods for delivery of macromolecular drugs to tumors. We have assessed a mouse model of leukemia caused by Mll-Enl protein fusion as a preclinical situation in which myeloid-lineage leukemia results from de novo occurrence of chromosomal translocations between Mll and Enl genes. Here, we show that the mouse leukemias respond to cytosine arabinoside, a frontline treatment for human leukemia. The observations show that the myeloid cells are susceptible to the drug and the mice undergo a remission that comprises a reduction of the myeloid population of cells and recovery of the lymphoid population. This translocator model should therefore prove useful for future drug assessments against the recurrent mixed-lineage leukemia-associated translocations.

Performance studies of a conical nozzle designed for the macromolecular skin delivery

Human skin is an attractive site for the delivery of protein and peptide-based macromolecular drugs for the treatment of topical and systemic diseases as well as for DNA immunisation. However, the delivery of those macromolecules in or across the skin is undesirably limited due to its permeation property. To expand the number of macromolecules to be delivered to specific targeting tissue/cells, a unique biomedical device, the handheld powdered injection system, has been developed. It is a novel transdermal technology that disposes needles (and syringes), circumvents the need for refrigeration (of vaccines) and has the potential to revolutionise the treatment and prevention of major diseases. To further underwrite device characteristics, in this paper, an advanced computational fluid dynamics technology is utilised to model gas, particle dynamics and gas–particle-target interaction. The statistical analyses show that the microparticles can achieve a mean velocity of 704 m/s representative of intracellular macromolecular deliveries. Knowledge on the gas and particle dynamics can be applied to design effective and efficient handheld powdered delivery systems.

Chitosan-Polycarbophil Interpolyelectrolyte Complex as an Excipient for Bioadhesive Matrix Systems to Control Macromolecular Drug Delivery

The in vitro performance of monolithic matrix systems containing the interpolyelectrolyte complex between chitosan and polycarbophil as excipient was evaluated in terms of their swelling, bioadhesive, and drug release properties. The different matrix systems showed excellent swelling properties without erosion, except for the formulation containing the highest quantity chitosan-polycarbophil complex that exhibited surface erosion in addition to swelling. All the different matrix systems exhibited significantly higher bioadhesive properties than the control group. Furthermore, they showed controlled insulin release without an initial burst release effect. However, only the matrix system that exhibited surface erosion in combination with swelling approached zero-order release.

Effect of pH on the drug release rate from a new polymer–drug conjugate system

AbstractThe corresponding N‐hydroximide and N‐methyl‐N‐hydroximide of poly[ethylene‐alt‐(maleic anhydride)] (weight average molecular weight (Mw) of 100–500 g mol−1) were prepared as a new oral drug delivery system. Syntheses of N‐hydroximide and N‐methylhydroxamic acid of poly[ethylene‐alt‐(maleic anhydride)] were carried out by chemical modification of polymer with hydroxylamine and N‐methylhydroxylamine, respectively, to give water‐soluble polymers. These activated polymers were immobilized with ketoprofen in the presence of dicyclohexylcarbodiimide to give the corresponding water‐insoluble ketoprofen conjugates. All products were characterized by elemental analysis as well as Fourier transform infrared and 1H NMR spectra. In vitro release of ketoprofen was studied by measuring UV absorption at λmax = 260 nm as a function of time. This study demonstrated the potential use of N‐hydroximide and N‐methyl‐N‐hydroxamic acid of poly[ethylene‐alt‐(maleic anhydride)] as a drug delivery system. Controlled release was studied at different pH values and at different temperatures. At physiological temperature, the amount of drug released increased with increasing pH. The copolymer‐drug adducts released the drug very slowly at the low pH found in the stomach thus protecting the drug from the action of high acid conditions and resident digestive enzymes. These N‐hydroxamic acid polymer‐drug conjugates were found to be potentially useful in the delivery of macromolecular drugs to targeted sites in the lower gastrointestinal tract and the colon area. Copyright © 2007 Society of Chemical Industry

Chitosan/cyclodextrin nanoparticles as macromolecular drug delivery system

The aim of this study was to generate a new type of nanoparticles made of chitosan (CS) and carboxymethyl-β-cyclodextrin (CM-β-CD) and to evaluate their potential for the association and delivery of macromolecular drugs. CS and CM-β-CD or mixtures of CM-β-CD/tripolyphosphate (TPP) were processed to nanoparticles via the ionotropic gelation technique. The resulting nanoparticles were in the size range of 231–383 nm and showed a positive zeta potential ranging from +20.6 to +39.7 mV. These nanoparticles were stable in simulated intestinal fluid pH 6.8 at 37 °C for at least 4 h. Elemental analysis studies revealed the actual integration of CM-β-CD to CS nanoparticles. Insulin and heparin used as macromolecular model drugs, could be incorporated into the different nanocarriers with association efficiencies of 85.5–93.3 and 69.3–70.6%, respectively. The association of these compounds led to an increase of the size of the nanoparticles (366–613 nm), with no significant modification of their zeta potentials (+23.3 to +37.1 mV). The release profiles of the associated macromolecules were highly dependent on the type of molecule and its interaction with the nanomatrix: insulin was very fast released (84–97% insulin within 15 min) whereas heparin remained highly associated to the nanoparticles for several hours (8.3–9.1% heparin within 8 h). In summary, CS-CD (cyclodextrin) nanoparticles may be considered as nanocarriers for the fast or slow delivery of macromolecules.

Studies on the Mechanism of Interaction of a Bioresponsive Endosomolytic Polyamidoamine with Interfaces. 1. Micelles as Model Surfaces

Polymers are appealing as pH-responsive elements of multicomponent systems designed to promote cytosolic delivery of macromolecular drugs (including proteins and genes), but so far the delivery efficiency achieved has been relatively modest. Therefore, the aim of this study was to apply several physicochemical techniques that are well established in the colloid field (surface tension measurements, small-angle neutron scattering (SANS), and electron paramagnetic resonance (EPR)) to probe the mechanism of endosomolytic polymer-surface interaction over the pH range 7.4 to 5.5 using the poly(amidoamine) (PAA) ISA23 x HCl and a series of "model" micelle surfaces. These micellar models were chosen to represent increasing complexity from simple, single surfactant sodium dodecylsulfate (SDS) micelles, surfactant mixtures containing bulky malono-bis-N-methylglucamide headgroups, or highly extended ethylene oxide headgroups. Spherical micelles composed of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) were also used. Changes in the onset of micellization, micelle surface fluidity, and in selected cases, the overall micelle shape and size were all quantified as a function of pH in the presence and absence of ISA23 x HCl. This amphoteric PAA is negatively charged at pH 7.4 and becomes gradually more protonated on exposure to lower pH values representative of the endosomal-lysosomal pathway. As expected, the strength of polymer interaction with anionic micelles increased with a decrease in pH, while for cationic micelles the opposite was observed. Addition of bulky, nonionic surfactant headgroups led to weaker interactions. The observations from surface tension and SANS studies showed a complex pattern of interaction with both an electrostatic and hydrophobic component. Using EPR it was confirmed that ISA23 x HCl perturbed the micelle palisade layer leading to a decrease in fluidity of the interface with a lower degree of headgroup hydration, and a significant change in micelle morphology. Surprisingly, there was no interaction between ISA23 x HCl and globular micelles formed from lyso-PC (a more biologically relevant model), and this suggests that the PAA structure could be better optimized to promote rapid interaction with endosomal membranes at the physiologically relevant pH 6.5.