Pharmacokinetics and Brain Uptake of Biotinylated Basic Fibroblast Growth Factor Conjugated to a Blood-Brain Barrier Drug Delivery System

Human basic fibroblast growth factor (bFGF) is a potent neuroprotective agent. The clinical efficacy of this neurotrophin, however, is restricted by poor permeability across the blood-brain barrier (BBB). This study was designed to test the hypotheses that bFGF will retain its biological activity and have an enhanced BBB transport after re-formulation and conjugation to a BBB peptide drug delivery vector. The BBB delivery vector is comprised of a conjugate of streptavidin (SA) and the murine OX26 monoclonal antibody against the rat transferrin receptor, and the conjugate of biotinylated bFGF (bio-bFGF) bound to a vector is designated bio-bFGF/OX26-SA. A radioreceptor binding assay shows that the native bFGF, bio-bFGF, and bio-bFGF/OX26-SA conjugate have IC 50 values of 0.12, 0.40, and 0.56 nM, respectively. After an IV bolus injection to the rat, [125 I]-bio-bFGF is avidly taken up by peripheral organs, with low brain uptake at 60 min, 0.010 ± 0.004% of injected dose (ID)/g brain. By contrast, the brain uptake of the [125 I]-bio-bFGF/OX26-SA is increased 5-fold to 0.050 ± 0.011%ID/g, although the uptake of the conjugate by peripheral tissues was decreased relative to the unconjugated bio-bFGF. In conclusion, conjugation of bio-bFGF to a BBB drug delivery vector (a) causes only a minor decrease in affinity for the bFGF receptor, (b) decreases the peripheral organ uptake of the bFGF, and (c) increases the brain uptake of the neurotrophin. The re-formulation of bFGF to enable receptor-mediated transcytosis across the BBB may improve the therapeutic index of this neurotrophin as a neuroprotective agent.     

Transport of Human Recombinant Brain-Derived Neurotrophic Factor (BDNF) Through the Rat Blood−Brain Barrier in Vivo Using Vector-Mediated Peptide Drug Delivery

The blood–brain barrier (BBB) transport of brain-derived neurotrophic factor (BDNF) in anesthetized rats was examined in the present studies using vector-mediated peptide drug delivery. Following tritiation, the BDNF was biotinylated via a disulfide linker and was coupled to a covalent conjugate of neutral avidin (NLA), which binds the biotinylated peptide with a high affinity, and the murine OX26 monoclonal antibody to the rat transferrin receptor. Owing to the abundance of transferrin receptors on brain capillary endothelium, the OX26 monoclonal antibody undergoes receptor-mediated transcytosis through the BBB, and the NLA–OX26 conjugate transports biotinylated peptide therapeutics through the BBB. The present studies show that while unconjugated BDNF was not transported through the BBB in vivo, the conjugation of biotinylated BDNF to the NLA–OX26 vector resulted in a marked increase in the brain delivery of BDNF, as defined by measurements of the percentage of the injected dose (ID) delivered per gram of brain. Although BDNF was not transported through the BBB in vivo, this cationic peptide was avidly bound by isolated human brain capillaries via a low-affinity, high-capacity system that was inhibited by protamine and by serum protein binding of BDNF. In conclusion, these studies show that the delivery of unconjugated BDNF to brain is nil owing to the combined effects of negligible BBB transport and rapid systemic clearance of intravenous administered BDNF. The brain delivery of BDNF may be augmented by conjugation of BDNF to BBB drug delivery vectors, such as the NLA–OX26 conjugate.     

Combined Use of Carboxyl-Directed Protein Pegylation and Vector-Mediated Blood-Brain Barrier Drug Delivery System Optimizes Brain Uptake of Brain-Derived Neurotrophic Factor Following Intravenous Administration

Purpose. Peptide drug delivery to the brain requires optimization of (a) plasma pharmacokinetics and (b) blood-brain barrier (BBB) permeability. In the present studies, plasma pharmacokinetics are improved with protein pegylation and BBB transport is facilitated with the use of vector-mediated drug delivery using the OX26 monoclonal antibody (MAb) to the rat transferrin receptor, which undergoes receptor-mediated transcytosis through the BBB in vivo. Methods. A conjugate of OX26 and streptavidin (SA), designated OX26/SA, was prepared in parallel with the carboxyl-directed pegylation of brain-derived neurotrophic factor (BDNF). A novel bifunctional polyethyleneglycol (PEG) was used in which a hydrazide (Hz) was attached at one end and a biotin moiety was attached to the other end. This allowed for conjugation of BDNF-PEG-biotin to OX26/SA. Results. The brain uptake of BDNF-PEG-biotin was increased following conjugation to OX26/SA to a level of 0.144 ± 0.004% injected dose per g brain and a BBB permeability-surface area product of 2.0 ± 0.2 L/min/g. Conclusions. These studies demonstrate that peptide drug delivery to the brain can be achieved with advanced formulation of protein-based therapeutics. The formulation is intended to (a) minimize rapid systemic clearance of the peptide, and (b) allow for vector-mediated drug delivery through the BBB in vivo. Following this dual formulation, the brain uptake of a neurotrophin such as BDNF achieves a value that is approximately 2-fold greater than that of morphine, a neuroactive small molecule.     

Stability of the disulfide bond in an avidin-biotin linked chimeric peptide during in vivo transcytosis through brain endothelial cells

Drug delivery of potential neuropharmaceuticals with poor intrinsic permeability through the blood-brain barrier (BBB), such as peptides, is facilitated by coupling to a vector that undergoes receptor-mediated transcytosis through the endothelial cells of brain microvessels. When cleavable disulfide linkers are used in the synthesis of such "chimeric peptides", it is crucial that the S-S-bridge is stable during transcytosis. Cleavage within endothelial cells could result in sequestration of the drug moiety instead of passage through the BBB. In the present study the metabolically stable opioid peptide [3 H]DALDA ([3 H]Tyr-DArg-Phe-Lys-NH2 ) was used as a model drug. It was monobiotinylated with the cleavable biotin reagent sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate (NHS-SS-biotin) to obtain bio-[3 H]DALDA. The biotinylated peptide was then bound to a vector for brain delivery after intravenous injection in rats, a covalent conjugate of streptavidin and the transferrin receptor monoclonal antibody, OX26. Compared to peptide without vector, brain uptake of bio-[3 H]DALDA after was increased 18-fold to reach 0.12% of the injected dose per g tissue. Transcranial microdialysis was performed for 60 min after an intravenous bolus of chimeric peptide, followed by reverse phase HPLC of dialysate. Stability of the chimeric peptide during transport through the BBB into brain extracellular fluid was concluded from the absence of a peptide peak generated by disulfide cleavage.     

Brain Delivery of Biotin Bound to a Conjugate of Neutral Avidin and Cationized Human Albumin

The delivery of pharmaceuticals through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, may be facilitated by conjugation of therapeutics to brain drug delivery vectors. Since cationized albumin has been shown to undergo absorptive-mediated transcytosis through the BBB in vivo, cationized human serum albumin (cHSA) is a potential brain drug delivery vector in humans. Conjugation of biotinylated therapeutics to brain drug delivery vectors is facilitated by the preparation of vector/ avidin conjugates. Therefore, the present studies describe the preparation of a cHSA-avidin conjugate and the delivery of ³H-biotin bound to this conjugate through the BBB in vivo in anesthetized rats. Since the cationic nature of avidin (AV) accelerates the removal of avidin-based conjugates from blood in vivo, the present studies also describe the preparation and the pharmacokinetics of ³H-biotin bound to a conjugate of cHSA and neutral avidin (NLA). The bifunctional nature of the conjugate was retained: the cHSA/ NLA conjugate contained 2.8 to 6.8 biotin binding sites per conjugate, and the BBB permeability-surface area (PS) product for ³H-biotin bound to cHSA/NLA was at least 7-fold greater than the BBB PS product for ³H-biotin bound to a conjugate of NLA and native HSA (nHSA). The systemic clearance of the cHSA conjugate was reduced 10-fold by the use of NLA as opposed to AV. The increased area under the plasma concentration curve (AUC) of the cHSA-NLA conjugate correlated with an increase in brain delivery of ³H-biotin as compared to the brain delivery achieved with the cHSA/AV conjugate. In conclusion, these studies demonstrate that cHSA serves as a brain drug delivery vector and that the use of neutral forms of avidin increases the plasma AUC of the conjugate and enhances the brain delivery of biotin.     

Delivery of peptides and proteins through the blood-brain barrier

Peptide and protein therapeutics are generally excluded from transport from blood to brain, owing to the negligible permeability to these drugs of the brain capillary endothelial wall, which makes up the blood—brain barrier (BBB) in vivo. However, peptides or protein therapeutics may be delivered to brain with the use of the chimeric peptide strategy for peptide drug delivery. Chimeric peptides are formed when a non-transportable peptide therapeutic is coupled to a BBB drug transport vector. Transport vectors are proteins such as cationized albumin, or the OX26 monoclonal antibody to the transferrin receptor; these proteins undergo absorptive-mediated and receptor-mediated transcytosis through the BBB, respectively. In addition to vector development, another important element of the chimeric peptide strategy is the design of strategies for coupling drugs to the vector that give high efficiency coupling and result in the liberation of biologically active peptide following cleavage of the bond linking the therapeutic and the transport vector. The avidin/biotin system has been recently shown to be advantageous in fulfilling these criteria for successful linker strategies. The use of the OX26 monoclonal antibody, the use of the avidin/biotin system as a linker strategy, and the design of a vasoactive intestinal peptide (VIP) analogue that is suitable for monobiotinylation and retention of biologic activity following cleavage, allowed for the recent demonstration of in vivo pharmacologic effects in brain following the systemic administration of relatively low doses (12 μg/kg) of neuropeptide.     

Delivery of peptides and proteins through the blood-brain barrier

Peptide and protein therapeutics are generally excluded from transport from blood to brain, owing to the negligible permeability of these drugs to the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. However, peptides or protein therapeutics may be delivered to the brain with the use of the chimeric peptide strategy for peptide drug delivery. Chimeric peptides are formed when a non-transportable peptide therapeutic is coupled to a BBB drug transport vector. Transport vectors are proteins such as cationized albumin, or the OX26 monoclonal antibody to the transferrin receptor; these proteins undergo absorptive-mediated and receptor-mediated transcytosis through the BBB, respectively. In addition to vector development, another important element of the chimeric peptide strategy is the design of strategies for coupling drugs to the vector that give high efficiency coupling and result in the liberation of biologically active peptides following cleavage of the bond linking the therapeutic and the transport vector. The avidin/biotin system has been recently shown to be advantageous in fulfilling these criteria for successful linker strategies. The use of the OX26 monoclonal antibody, the use of the avidin/biotin system as a linker strategy, and the design of a vasoactive intestinal peptide (VIP) analogue that is suitable for monobiotinylation and retention of biologic activity following cleavage, allowed for the recent demonstration of in vivo pharmacologic effects in brain following the systemic administration of relatively low doses (12 microg/kg) of neuropeptide.     

Brain drug targeting and gene technologies.

Brain drug targeting technology is based on the application of four gene technologies that enable the delivery of drugs or genes across the blood-brain barrier (BBB) in vivo. I) Genetic engineering is used to produce humanized monoclonal antibodies that target endogenous BBB transporters and act as vectors for delivery of drugs or genes to the human brain. The conjugate of a neurotherapeutic and a BBB transport vector is called a chimeric peptide. Epidermal growth factor chimeric peptides have been used for neuroimaging of brain cancer. Brain-derived neutrophic factor chimeric peptides have marked neuroprotective effects in brain stroke models. II) Imaging gene expression in the brain in vivo is possible with sequence-specific antisense radiopharmaceuticals, which are conjugated to BBB drug targeting vectors. III) Brain gene targeting technology enables widespread expression of an exogenous gene throughout the central nervous system following an intravenous injection of a non-viral therapeutic gene. IV) A BBB genomics program enables the future discovery of novel transport systems expressed at the BBB. These transporters may be carrier-mediated transport systems, active efflux transporters, or receptor-mediated transcytosis systems. The future discovery of novel BBB transport systems and the application of brain drug targeting technology will enable the delivery to the brain of virtually any neurotherapeutic, including small molecules, large molecules and gene medicines.     

Drug targeting to the brain using avidin-biotin technology in the mouse; (blood-brain barrier, monoclonal antibody, transferrin receptor, Alzheimer's disease)

A beta1-40 peptide radiopharmaceuticals could be used to image A beta brain amyloid in transgenic mouse models of Alzheimer's disease should the A beta peptide radiopharmaceutical be made transportable through the blood-brain barrier (BBB) in vivo. The present studies used the RI7-217 rat monoclonal antibody to the mouse transferrin receptor as a BBB drug targeting vector for the delivery to brain of A beta1-40 radiolabeled with either 125-Iodine or 111-Indium. The A beta peptide radiopharmaceutical is conjugated to the RI7 MAb using avidin biotin technology, wherein the A beta1-40 peptide radiopharmaceutical is monobiotinylated (bio) and bound to a conjugate of the RI7 MAb and streptavidin (SA). The [125 I]-bio-A beta1-40 or the [111 In]-bio-A beta1-40 either free or bound to the RI7/SA conjugate was injected intravenously into anesthetized adult mice and plasma pharmacokinetics and organ uptake were measured over the next 60 minutes. The A beta1-40 peptide radiopharmaceutical radiolabeled with 111-Indium was the preferred formulation, compared to peptide labeled with 125-Iodine, because there was a greater metabolic stability and reduced artifactual organ uptake of metabolites associated with the use of the 111-Indium nuclide. However, biotinylated A beta1-40 peptide radiopharmaceuticals conjugated to the RI7/SA brain drug targeting system were metabolically unstable in mice in vivo owing to active biotinidase activity. Future work involving brain drug targeting in mice that utilizes avidin biotin technology will need to incorporate biotin analogues that are resistant to biotinidase.     

Neurotrophins,neuroprotection and the blood-brain barrier

Small molecule drugs have not been effective neuroprotective agents in either the acute treatment of stroke or the chronic treatment of neurodegeneration. Thus, it is time to consider large molecule drugs such as recombinant neurotrophins. However, like many of the small molecules, neurotrophins do not cross the brain capillary endothelial wall, which forms the blood-brain barrier (BBB). Neurotrophins can be made transportable across the BBB by using chimeric peptide brain drug targeting technology, in which a neurotrophin is reformulated by fusion to a transport vector. The latter is a peptide or peptidomimetic monoclonal antibody that undergoes receptor-mediated transcytosis through the BBB, and acts as a 'molecular Trojan horse'. Neurotrophin chimeric peptides are highly neuroprotective following delayed intravenous administration, in both global and focal brain ischemia.     

Uptake of ANG1005, A Novel Paclitaxel Derivative, Through the Blood-Brain Barrier into Brain and Experimental Brain Metastases of Breast Cancer

Purpose

We evaluated the uptake of angiopep-2 paclitaxel conjugate, ANG1005, into brain and brain metastases of breast cancer in rodents. Most anticancer drugs show poor delivery to brain tumors due to limited transport across the blood-brain barrier (BBB). To overcome this, a 19-amino acid peptide (angiopep-2) was developed that binds to low density lipoprotein receptor-related protein (LRP) receptors at the BBB and has the potential to deliver drugs to brain by receptor-mediated transport.

Methods

The transfer coefficient (K_in) for brain influx was measured by in situ rat brain perfusion. Drug distribution was determined at 30 min after i.v. injection in mice bearing intracerebral MDA-MB-231BR metastases of breast cancer.

Results

The BBB K_in for ¹²⁵I-ANG1005 uptake (7.3?±?0.2?×?10^-3 mL/s/g) exceeded that for ³H-paclitaxel (8.5?±?0.5?×?10^-5) by 86-fold. Over 70% of ¹²⁵I-ANG1005 tracer stayed in brain after capillary depletion or vascular washout. Brain ¹²⁵I-ANG1005 uptake was reduced by unlabeled angiopep-2 vector and by LRP ligands, consistent with receptor transport. In vivo uptake of ¹²⁵I-ANG1005 into vascularly corrected brain and brain metastases exceeded that of ¹⁴C-paclitaxel by 4–54-fold.

Conclusions

The results demonstrate that ANG1005 shows significantly improved delivery to brain and brain metastases of breast cancer compared to free paclitaxel.     

Brain delivery of biotinylated NGF bounded to an avidin-transferrin conjugate

Recent study showed that transferrin receptors were concentrated on the plasma membrane of brain endothelia cells and mediated transcytosis of transferrin (Tf) through the blood-brain barrier (BBB). This property allows the transferrin to act as the brain drug transporter vector. The present investigation examined the pharmacokinetic behavior of nerve growth factor (NGF), which was conjugated to transferrin by the avidin/biotin technology, especially its brain-uptake efficiency. The area under the plasma concentration curve and the mean residence time were not significantly different for either bio-NGF or bio-NGF/AV-Tf. At the first hour after single intravenous injection, the BBB permeability surface area product of bio-NGF/AV-Tf was 0.77 microliter/min/g; it was about 8-fold higher than that of bio-NGF, and equal to that of AV-OX26. The delivery of bio-NGF/AV-Tf to brain was 0.075% of injected dose per gram brain, and it was 5-fold higher than that of bio-NGF, and 2-fold higher than that of AV-OX26. In summary, these studies demonstrated that the use of Tf as brain drug delivery vector was as effective in transporting biotinylated therapeutics as OX26, and avoided the disadvantages of its antigenicity.     

Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway

The membrane transferrin receptor-mediated endocytosis or internalization of the complex of transferrin bound iron and the transferrin receptor is the major route of cellular iron uptake. This efficient cellular uptake pathway has been exploited for the site-specific delivery not only of anticancer drugs and proteins, but also of therapeutic genes into proliferating malignant cells that overexpress the transferrin receptors. This is achieved either chemically by conjugation of transferrin with therapeutic drugs, proteins, or genetically by infusion of therapeutic peptides or proteins into the structure of transferrin. The resulting conjugates significantly improve the cytotoxicity and selectivity of the drugs. The coupling of DNA to transferrin via a polycation or liposome serves as a potential alternative to viral vector for gene therapy. Moreover, the OX26 monoclonal antibody against the rat transferrin receptor offers great promise in the delivery of therapeutic agents across the blood-brain barrier to the brain.     

Current strategies for targeted delivery of bio-active drug molecules in the treatment of brain tumor

Brain tumor is one of the most challenging diseases to treat. The major obstacle in the specific drug delivery to brain is blood–brain barrier (BBB). Mostly available anti-cancer drugs are large hydrophobic molecules which have limited permeability via BBB. Therefore, it is clear that the protective barriers confining the passage of the foreign particles into the brain are the main impediment for the brain drug delivery. Hence, the major challenge in drug development and delivery for the neurological diseases is to design non-invasive nanocarrier systems that can assist controlled and targeted drug delivery to the specific regions of the brain. In this review article, our major focus to treat brain tumor by study numerous strategies includes intracerebral implants, BBB disruption, intraventricular infusion, convection-enhanced delivery, intra-arterial drug delivery, intrathecal drug delivery, injection, catheters, pumps, microdialysis, RNA interference, antisense therapy, gene therapy, monoclonal/cationic antibodies conjugate, endogenous transporters, lipophilic analogues, prodrugs, efflux transporters, direct conjugation of antitumor drugs, direct targeting of liposomes, nanoparticles, solid–lipid nanoparticles, polymeric micelles, dendrimers and albumin-based drug carriers.     

Pharmacological actions of nerve growth factor-transferrin conjugate on the central nervous system.

Transferrin (Tf) undergoes receptor-mediated transport through the blood-brain barrier in vivo. This property allows transferrin to act as a vector for drug transport to the brain. The present investigation examines both the profile of brain delivery of nerve growth factor (NGF)-transferrin conjugate, and its therapeutic effects on CNS neurodegeneration, which affect locomotion and memory. Delivery of NGF-Tf conjugate to the brain was measured and found to be 0.075% of the injected dose per gram of brain, which is 5-fold higher than that of biotin-NGF. The increased delivery using the NGF-Tf conjugate can be attributed to an increased BBB permeability surface area product, which is about 8-fold higher than that of biotin-NGF. Intravenous injection of biotin-NGF/Tf-avidin conjugate significantly increased neuronal survival in the substantia nigra of a Parkinson's disease mouse model. In addition, this conjugate also improved recognition and memory in Alzheimer's disease rat model. In summary, these results demonstrate that using transferrin as a delivery vector is effective in targeting NGF to the central nervous system (CNS), and may optimize the therapy of age-related neurodegenerative diseases.     

Endocytosis and transcytosis of an immunoliposome-based brain drug delivery system

Immunoliposomes conjugated with the OX26 monoclonal antibody to the rat transferrin receptor can be used for brain delivery of small molecules. In the present study the uptake of OX26-immunoliposomes by target cells as well as their transcytosis across the blood-brain barrier was investigated. Microscopy of RG2 rat glioma cells incubated with fluorescence labeled OX26-immunoliposomes revealed intracellular co-localization of liposomal cargo, the liposomal membrane bilayer and the OX26 monoclonal antibody. The distinct particulate staining pattern was indicative for accumulation of OX26-immunoliposomes within endosomal or lysosomal compartments. Prolonged incubations demonstrated endosomal release of the liposomal cargo propidium iodide to the cytoplasm. A maximum of 50% of propidium iodide was released from the endosomal compartment after 24 hours of incubation. Transcytosis was studied using an in vitro model of the blood-brain barrier consisting of immortalized RBE4 rat brain endothelial cells. OX26-immunoliposomes did permeate across the RBE4 cell monolayer and showed a permeability coefficient of P(app) = 1.6 x 10(-5) ml/s. Transport was inhibited at low temperature, by competition with free OX26 or by exchanging the OX26 monoclonal antibody for an unspecific isotype antibody. Transcytosis of OX26-immunolipsomes was confirmed in vivo by the brain perfusion and capillary depletion technique. OX26-immunoliposomes were detected within the post-vascular compartment of brain parenchyma (PS product = 2.4 microl/g/min.) and were not associated with the brain microvasculature.     

Conjugation with L-Glutamate for in vivo brain drug delivery

In vitro studies have shown that conjugation of a model compound [p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivo brain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5,7-dichlorokynurenic acid (DCKA) and D-kyotorphin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.     

Production of Soluble and Active Transferrin Receptor-Targeting Single-Chain Antibody using Saccharomyces cerevisiae

Purpose This study describes the soluble production, purification, and functional testing of an anti-transferrin receptor single-chain antibody (OX26 scFv) using the yeast Saccharomyces cerevisiae. Methods The yeast secretion apparatus was optimized by modulating expression temperature, the folding environment of the endoplasmic reticulum, and gene dosage. Secreted scFv was purified using immobilized metal affinity chromatography, and tested for binding and internalization into the RBE4 rat brain endothelial cell line. Results Secretion of OX26 scFv was optimal when expression was induced at 20°C. Co-overexpression of heavy chain binding protein and protein disulfide isomerase elevated scFv expression levels by 10.4 ± 0.3-fold. Optimization of scFv gene dosage increased secretion by 7.1 ± 0.2-fold, but the overall benefits of binding protein and protein disulfide isomerase overexpression were diminished. Purified OX26 scFv yields of 0.5 mg/L secreted protein were achieved, and the scFv was actively internalized into RBE4 cells with a pattern similar to that observed with intact OX26 monoclonal antibody. Conclusions The optimized S. cerevisiae expression system is amenable to production of soluble and active brain targeting OX26 scFv, and the yeast-produced scFv has potential for the targeting and delivery of small molecules, proteins, or drug carriers across the blood–brain barrier(BBB).     

By-passing of P-glycoprotein using immunoliposomes

The over-expression of MDR1 P-glycoprotein has been associated with the development of multidrug-resistance in cancer cells. Methods used to overcome multidrug-resistance often involve the co-administration of inhibitors of P-glycoprotein. Here, we test the hypothesis that an immunoliposome-based drug delivery system may be used as an alternative approach to overcome multidrug-resistance since immunoliposomes penetrate target cells by receptor-mediated endocytosis which allows to by-pass membrane-associated P-glycoprotein. Targeting of immunoliposomes was achieved by the use of an anti-transferrin receptor monoclonal antibody (OX26 mAb). Incorporation of radiolabelled digoxin within OX26-immunoliposomes enhanced cellular uptake of digoxin by a factor of 25 in immortalised RBE4 rat brain capillary endothelial cells. Uptake of liposomal digoxin was insensitive to ritonavir, a P-glycoprotein inhibitor, and was reduced in presence of increasing free concentrations of OX26 mAb or nocodazole, a reversible inhibitor of endocytosis. In contrast, uptake of free digoxin was enhanced by a factor of 1.8 in presence of ritonavir and was insensitive to OX26 mAb or nocodazole. Cellular uptake and intracellular accumulation of liposomal digoxin (55% internalisation within 30 min) was demonstrated by acid wash of the cells and was confirmed by confocal microscopy studies. Endosomal release to the cytosol of propidium iodide loaded immunoliposomes was shown. These in vitro studies suggest that immunoliposome-based drug delivery systems can be used to by-pass P-glycoprotein and thus deliver drugs to the cytosol of a target cell.     

By-passing of P-glycoprotein Using Immunoliposomes

The over-expression of MDR1 P-glycoprotein has been associated with the development of multidrug-resistance in cancer cells. Methods used to overcome multidrug-resistance often involve the co-administration of inhibitors of P-glycoprotein. Here, we test the hypothesis that an immunoliposome-based drug delivery system may be used as an alternative approach to overcome multidrug-resistance since immunoliposomes penetrate target cells by receptor-mediated endocytosis which allows to by-pass membrane-associated P-glycoprotein. Targeting of immunoliposomes was achieved by the use of an anti-transferrin receptor monoclonal antibody (OX26 mAb). Incorporation of radiolabelled digoxin within OX26-immunoliposomes enhanced cellular uptake of digoxin by a factor of 25 in immortalised RBE4 rat brain capillary endothelial cells. Uptake of liposomal digoxin was insensitive to ritonavir, a P-glycoprotein inhibitor, and was reduced in presence of increasing free concentrations of OX26 mAb or nocodazole, a reversible inhibitor of endocytosis. In contrast, uptake of free digoxin was enhanced by a factor of 1.8 in presence of ritonavir and was insensitive to OX26 mAb or nocodazole. Cellular uptake and intracellular accumulation of liposomal digoxin (55% internalisation within 30 min) was demonstrated by acid wash of the cells and was confirmed by confocal microscopy studies. Endosomal release to the cytosol of propidium iodide loaded immunoliposomes was shown. These in vitro studies suggest that immunoliposome-based drug delivery systems can be used to by-pass P-glycoprotein and thus deliver drugs to the cytosol of a target cell.