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Biomedical subjects

N Murthy

Publications and source records attributed to N Murthy.

17 recordsLinked to original sources
PubMed2005

'Smart' delivery systems for biomolecular therapeutics.

OBJECTIVE: There is a strong need for drug delivery systems that can deliver biological signals from biomaterials and tissue engineering scaffolds, and a particular need for new delivery systems that can efficiently deliver biomolecules to intracellular targets. Viruses and pathogens have evolved potent molecular machinery that sense the lowered pH gradient of the endosomal compartment and become activated to destabilize the endosomal membrane, thereby enhancing protein or DNA transport to the cytoplasmic compartment. A key feature of many of these biological delivery systems is that they are reversible, so that the delivery systems are not directly toxic. These delivery systems have the ability to change their structural and functional properties and thus display remarkable 'smart' material properties. The objective of this presentation is to review the initial development of smart polymeric carriers that mimic these biological delivery systems and combine similar pH-sensitive, membrane-destabilizing activity for the delivery of therapeutic biomolecules. DESIGN: We have developed new 'smart' polymeric carriers to more effectively deliver and broaden the available types of biomolecular therapeutics. The polymers are hydrophilic and stealth-like at physiological pH, but become membrane-destabilizing after uptake into the endosomal compartment where they enhance the release of therapeutic cargo into the cytoplasm. They can be designed to provide a range of pH profiles and membrane-destabilizing activities, allowing their molecular properties to be matched to specific drugs and loading ranges. A versatile set of linker chemistries is available to provide degradable conjugation sites for proteins, nucleic acids, and/or targeting moieties. RESULTS: The physical properties of several pH-responsive polymers were examined. The activity and pH profile can be manipulated by controlling the length of hydrophobic alkyl segments. The delivery of poly(propyl acrylic acid) (PPAA)-containing lipoplexes significantly enhanced wound healing through the interconnected effects of altered extracellular matrix organization and greater vascularization. PPAA has also been shown to enhance cytoplasmic delivery of a model protein therapeutic. Polymeric carriers displaying pH-sensitive, membrane-destabilizing activity were also examined. The pH profile is controlled by the choice of the alkylacrylic acid monomer and by the ratio of the carboxylate-containing alkylacrylic acid monomer to alkylacrylate monomer. The membrane destabilizing activity is controlled by the lengths of the alkyl segment on the alkylacrylic acid monomer and the alkylacrylate monomer, as well as by their ratio in the final polymer chains. CONCLUSION: The molecular mechanisms that proteins use to sense and destabilize provide interesting paradigms for the development of new polymeric delivery systems that mimic biological strategies for promoting the intracellular delivery of biomolecular drugs. The key feature of these polymers is their ability to directly enhance the intracellular delivery of proteins and DNA, by destabilizing biological membranes in response to vesicular compartment pH changes. The ability to deliver a wide variety of protein and nucleic acid drugs to intracellular compartments from tissue engineering and regenerative scaffolds could greatly enhance control of important processes such as inflammation, angiogenesis, and biomineralization.

Biocompatible Materials↗
PubMed2000

Founder's Award, Society for Biomaterials. Sixth World Biomaterials Congress 2000, Kamuela, HI,May 15-20, 2000. Really smart bioconjugates of smart polymers and receptor proteins.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Acrylamides↗
PubMed2000

Molecular engineering of proteins and polymers for targeting and intracellular delivery of therapeutics.

There are many protein and DNA based therapeutics under development in the biotechnology and pharmaceutical industries. Key delivery challenges remain before many of these biomolecular therapeutics reach the clinic. Two important barriers are the effective targeting of drugs to specific tissues and cells and the subsequent intracellular delivery to appropriate cellular compartments. In this review, we summarize protein engineering work aimed at improving the stability and refolding efficiency of antibody fragments used in targeting, and at constructing new streptavidin variants which may offer improved performance in pre-targeting delivery strategies. In addition, we review recent work with pH-responsive polymers that mimic the membrane disruptive properties of viruses and toxins. These polymers could serve as alternatives to fusogenic peptides in gene therapy formulations and to enhance the intracellular delivery of protein therapeutics that function in the cytoplasm.

Antibodies↗
PubMed1999

The design and synthesis of polymers for eukaryotic membrane disruption.

The intracellular trafficking of drugs is critical to the efficacy of drugs that are susceptible to attack by lysosomal enzymes. It is therefore an important goal to design and synthesize molecules which can enhance the transport of endocytosed drugs from the endosomal compartments to the cytoplasm. The pH of an endosome is lower than that of the cytosol by one to two pH units, depending on the stage of endosomal development. This pH gradient is a key factor in the design of membrane-disruptive polymers which could enhance the endosomal release of drugs. Such polymers should disrupt lipid bilayer membranes at pH 6.5 and below, but should be non-lytic at pH 7.4. We have designed and synthesized pH-sensitive synthetic polymers which efficiently disrupt red blood cells within a sharply defined pH range. One of these polymers, poly(ethyl acrylic acid) (PEAAc) has been previously shown to disrupt synthetic vesicles in a pH-dependent fashion [6]. PEAAc hemolyzes red blood cells with an activity of 10(7) molecules per red blood cell, which is as efficient on a molar basis as the peptide melittin. The mechanism of RBC hemolysis by PEAAc is consistent with the colloid osmotic mechanism. PEAAc's hemolytic activity rises rapidly as the pH decreases from 6.3 to 5.0, and there is no hemolytic activity at pH 7.4. A related polymer, poly(propyl acrylic acid) (PPAAc), was synthesized to test whether making the pendant alkyl group more hydrophobic by adding one methylene group would increase the hemolytic activity. PPAAc was found to disrupt red blood cells 15 times more efficiently than PEAAc at pH 6.1. PPAAc was also not active at pH 7.4 and displayed a pH-dependent hemolysis that was shifted toward higher pH's. Random 1:1 copolymers of ethyl acrylate (EA) and acrylic acid (AAc) (which contain random -COOH and -C(2)H(5) groups that are present and regularly repeat in PEAAc) also displayed significant hemolytic activity, with an efficiency close to PEAAc. These results demonstrate that pH-sensitive synthetic polymers can be molecularly engineered to efficiently disrupt eukaryotic membranes within defined and narrow pH ranges. Thus, these polymers might serve as endosomal disruptive agents with specificities for early or late endosomes.

Cell Membrane↗
PubMed1998

The replication inhibition activity of the WT1 tumor suppressor protein resides in its N-terminal 298 amino acid region, and does not require specific binding of the protein to the replication origin sequence.

Using the simian virus 40 replication origin as the model, it was reported previously that the Wilms' tumor suppressor protein WT1 can inhibit DNA replication. In the present study, we found that a hybrid protein (termed GAL4-WT1AE Z) consisting of the DNA-binding domain of the yeast transcription factor GAL4 fused in-frame with the N-terminal 298 amino acid (aa) transcriptional regulatory region of WT1 retained the ability to inhibit replication. The hybrid protein and the full-length WT1 inhibited replication without regard to the presence or absence of their binding sites in the replication origin region, indicating that inhibition of replication by the proteins does not require their specific binding to the origin region. The inhibition efficiency of the hybrid protein was the same as that of WT1, indicating that the replication inhibition activity resides in the N-terminal 298 aa region, and that the C-terminal zinc finger-containing DNA-binding domain of WT1 is functionally dispensable for this effect.

Amino Acid Sequence↗
PubMed1997

Systemic and mucosal immune response to polio vaccination with additional dose in newborn period.

Administration of supplementary dose of oral polio vaccine (OPV) during neonatal period is recommended by WHO in countries like India, where host response to the regular to the regular three-dose schedule is not satisfactory and poliomyelitis continues to be a problem. The efficacy of this dose +3, and +5 doses of OPV in terms of seropositivity, seroconversion, systemic, and mucosal antibody responses were measured in 51 infants in a follow-up study from birth to 30 weeks. Administration of the additional dose in the newborn period significantly improved seropositivity and seroconversion rates compared to the conventional 3 or 5 dose schedules. Systemic antibody titres improved with each dose of the vaccine and 40-60 per cent of infants had > 1:128 titres to the three types of polioviruses that could prevent re-infection of the gut and 50 per cent of them had mucosal antibodies as evidenced by specific IgA in nasopharyngeal secretions. Therefore, administration of OPV in the neonatal period should be made compulsory in countries where poliomyelitis still continues to be a problem.

Antibodies, Viral↗
PubMed1995

A direct ELISA technique to detect antibodies against polioviruses.

A direct ELISA test was developed to detect circulating antibodies specific to poliovirus types 1, 2 and 3. Specificity of the test was established by using known polio negative and positive sera. The assay was compared with the standard microneutralization test using sera from polio and non polio cases vaccinated and unvaccinated subjects and standard positive sera obtained from WHO/EPI (Geneva). The reproducibility of the results was tested using different batches of the antigen prepared from Sabin strain of poliovirus supplied by ERC Bombay and from Sabin strain of virus obtained from WHO/EPI. ELISA was found to be as sensitive as microneutralization test in detecting seronegatives and was found to be specific to polio by giving negative results with non polio cases. ELISA is thus a rapid and simple method that may be useful for mapping seroepidemiology of poliomyelitis and as a tool for effective surveillance of the disease by offering rapid diagnosis in acute cases.

Antibodies, Viral↗
PubMed1994

An outbreak of poliomyelitis in Andhra Pradesh (South India).

An outbreak of poliomyelitis that occurred in the year 1992 in Telangana region of Andhra Pradesh, South India was investigated to understand the reasons for persistence of poliomyelitis in the general population and for the outbreak in Andhra Pradesh in particular. The study comprised of a detailed investigation of epidemiological and clinical features, serology and vaccination status and a case control study to calculate vaccine efficacy by matched pair analysis. The outbreak occurred after a relative quiescence of 3 years. The age group of the patients ranged from 2 months to 5 years, 26.5% being infants and 70.2% being children between 1 and 5 years. The outbreak was mainly caused by Type 1 poliovirus. Vaccine efficacy was found to be 70%. Antibody response was not high in cases. Seventy six per cent of the children with poliomyelitis were unvaccinated. Ignorance of the mothers and family interference were the main causes for not vaccinating the children. The study indicates the need to increase the vaccination coverage and inclusion of children upto 5 years in the programme. Absence of vaccination is the major risk factor for the outbreak. The persistence of poliomyelitis in older children, low antibody response and suboptimal vaccine efficacy point out the problem of achieving control with OPV in tropical countries and suggest the need for alternate strategies. Better health education strategies need to be developed.

Child, Preschool↗
PubMed1991

Funding of health research in India.

An overview of financing of health research in India is presented based on data for the year 1987-88, collected from nearly 298 institutions involved in health research. Only 8 per cent of the funds was derived from foreign sources. Distribution of research funds was highly skewed. Research activity seemed to have concentrated in a few specialty institutes located in the four metropolitan cities. Research activities apparently matched the national health priorities, reproductive health being the most frequently reported area of research. The study concludes that since health is primarily a service sector, its research needs are not fully appreciated. As a result, research allocation in the health sector is lower, as compared to that for many other developmental sectors. The paper highlights some of the implications of this lopsided distribution to research capacity building in the country.

Humans↗
PubMedDate not supplied

A pH-sensitive polymer that enhances cationic lipid-mediated gene transfer.

The efficient release of nonviral gene carriers from endosomes is an important step for the successful delivery of DNA into the cell nucleus. A synthetic pH-sensitive anionic polymer, poly(propylacrylic acid) (PPAA), was designed to aid in endosomal escape of nonviral vectors and improve the transfection efficiencies with these vectors. Transfection of NIH3T3 fibroblasts with ternary physical mixtures of the cationic lipid DOTAP, pCMVbeta plasmid DNA, and PPAA showed marked enhancement of both gene expression levels and fraction of cells transfected compared to binary control mixtures of DOTAP and DNA. PPAA also significantly improved the serum-stability of DOTAP/DNA vectors. The DOTAP/DNA/PPAA vectors maintained high levels of transfection in media containing up to 50% serum. The striking enhancement of transfection efficiency with cationic lipid/DNA/PPAA mixtures, along with the enhanced serum-stability, suggests that PPAA may provide significant improvements for the in vivo intracellular delivery of drugs such as DNA, oligonucleotides, proteins, and peptides.

3T3 Cells↗
PubMedDate not supplied

Hemolytic activity of pH-responsive polymer-streptavidin bioconjugates.

Drug delivery systems that increase the rate and/or quantity of drug release to the cytoplasm are needed to enhance cytosolic delivery and to circumvent nonproductive cell trafficking routes. We have previously demonstrated that poly(2-ethylacrylic acid) (PEAAc) has pH-dependent hemolytic properties, and more recently, we have found that poly(2-propylacrylic acid) (PPAAc) displays even greater pH-responsive hemolytic activity than PEAAc at the acidic pHs of the early endosome. Thus, these polymers could potentially serve as endosomal releasing agents in immunotoxin therapies. In this paper, we have investigated whether the pH-dependent membrane disruptive activity of PPAAc is retained after binding to a protein. We did this by measuring the hemolytic activity of PPAAc-streptavidin model complexes with different protein to polymer stoichiometries. Biotin was conjugated to amine-terminated PPAAc, which was subsequently bound to streptavidin by biotin complexation. The ability of these samples to disrupt red blood cell membranes was investigated for a range of polymer concentrations, a range of pH values, and two polymer-to-streptavidin ratios of 3:1 and 1:1. The results demonstrate that (a) the PPAAc-streptavidin complex retains the ability to lyse the RBC lipid bilayers at low pHs, such as those existing in endosomes, and (b) the hemolytic ability of the PPAAc-streptavidin complex is similar to that of the free PPAAc.

Acrylates↗