Synthesis And Characterization Of PHBV Microspheres Loaded Biology Essay

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Rifampicin or rifampin is a bactericidal antibiotic drug of the rifamycin group. It is a semisynthetic compound derived from Amycolatopsis rifamycinica . Rifampicin inhibits DNA-dependent RNA polymerase in bacterial cells by binding its beta-subunit, thus preventing transcription to RNA and subsequent translation to proteins.

PHB is produced by micro-organisms like Alcaligenes eutrophus or Bacillus megaterium apparently in response to conditions of physiological stress. The polymer is primarily a product of carbon assimilation (from glucose or starch) and is employed by micro-organisms as a form of energy storage molecule to be metabolized when other common energy sources are not available. Microbial biosynthesis of PHB starts with the condensation of two molecules of acetyl-CoA to give acetoacetyl-CoA which is subsequently reduced to hydroxybutyryl-CoA. This hydroxybutyryl-CoA is then used as a monomer to polymerize PHB. Rifampicin was loaded in 5%PHBV polymer prepared in DCM and drug release was studied.

INTRODUCTION:

Nanotechnology is the cross disciplinary field, which involves the ability to to design and exploit the unique properties that emerge from man-made materials ranging in size from 1 to greater than 100 nm .Indeed, the physical and chemical properties of materials such as porosity,electrical conductivity, light emission, and magnetism can significantly improve or radically change as their size is scaled down to small clusters of atoms.

These advances are beginning to have a paradigm shifting impact not Least in experimental (eg: thermal tumor killing) and diagnostic oncology.Examples include super paramagnetic iron oxide nanocrystals, quantum dots (QD), inorganic nanoparticles, and compositenanoshells.The surfaces of these entities are amenable to modification with synthetic polymers(to afford long circulating properties) and for targeting ligands.The key problem with these technologies is toxicity.

Polymeric conjugates are used as vector systems for delivery of anti tumor agents. This polymer conjugates are targeted based on the over-expression of particular receptors present on the cancer cells.Copolymer nanovectors (eg: N-hydroxypropyl) methylacrylamide (HPMA) and Hyaluronan(HA) HPMA-HA serves as nanovectors for the delivery of chemotheraphy and radiotheraphy and also showspromise as agent to deliver. Polymer conjugates can be targeted to a specific site, can carry a multitude of biodegradable blood-pool contrasting agents for the use with tumour imaging technologies. Agents useful for cancer chemotherapeutics,radiotheraphy,or tumour imaging and their surfaces can be modified using different biological surface modifiers to enhance selectivity of localization of the nanovector.

Studies focusing on polymeric nanoparticles as nanovectors have included the use of poly(lactic-co-glycolic acid) (PLGA) and poly lactide(PLA) that can be selectively targeted against such surface receptors as transferrin receptor on breast tumor cells, as well as can be optimized to avoid biobarriers such as RES.In addition, polymeric nanoparticles can be altered to enhance the intracellular drug concentration once delivered. Polymeric nanoparticles are mutable for enhancement of the retention at the local tumor site and to combine antitumor agents with antiangiogenic strategies.

In addition to delivery of anti-tumor agents that are antiangiogenic, nanovectors such as PLGA,PLA as well as pegylated gelatin nanovectors,these have been improved by thiolation to optimize their time in circulation and to enhance delivery of payloads such as non-viral DNA for gene theraphy.

New polymeric vectors recently shown to have potential utility in cancer nanotechnology are bioconjugates composed of PLGA polymers and nucleic acid ligands defined as aptamers.These biovectors can be PEGylated and further modified with selectively targeted approaches directed against such proteins such as prostate specific antigen (PSA) which are present in abundance on the surface of prostatic tumor cells,these nanoparticle-aptamer bioconjugates represent a new class of polymeric nanoparticles with promise as multifunctional nanovectors.Polymeric aptamer bioconjugates are currently being evaluated for their optimization for use in a large number of systems that may lead to invivvo studies in the near future. Abraxane is the only example of a regulatory approved (FDA,USA) nanoparticle formulation for intravenous drug delivery in cancer patients.It is paclitaxel bound to albumin nanoparticles, with a mean diameter of 130 nm, for use in individuals with metastatic breast cancer who have failed combination chemothearaphy or relapse within 6 months of adjuvant chemotheraphy.This formulation overcomes poor solubility of paclitaxel in the blood and allow patients to receive 50% more paclitxel per dose over a 30 minute period.Albumin nanoparticles seem to interact with gp60 receptors present in the tumor blood vessels that transport the nanoparticles into tumor interstitial spaces by transcytosis, a process that partly contribute to the effectiveness of Abraxane.

Nanoparticles assembled from synthetic copolymers have also received much attention in cancer.One interesting example is doxorubicin-loaded poly(alkylcyanoacrylate)(PACA) nanoparticles.Invitrostudies have indicated that PACA nanoparticles can overcome drugresistance in tumorcells expressing multidrug-resistance-1-type efflux pumps. The mechanism of action is related to adherence of PACA nanoparticles to tumor cellplasmamembrane, which Initiates particle degradation and provides a concentration gradient for doxorubicin,and diffusion of doxorubicin across the plasmamembrane following formation of anion pair between the positively charged doxorubicin and the negatively charged cyanoacrylicacid (nananoparticle degradationproduct).

These observations clearly indicate that drug release and nanoparticleDegradation must occurs imultaneously ,yielding anappropriate size complex with correctphysi-cochemical properties for diffusion across the plasmamembrane. Polymer-based drug delivery systems also favorably alter the pharmacokinetics and biodistribution of conjugated drugs and accumulate in tumor interstitium following extrusion .Examples Include SMANCS (a conjugate of the polymer styrene- co - maleicacid / anhydride and neocarzinos - tatin for treatment of hepatocellular carcinoma), conjugates cytotoxic agents (e.g.,paclitaxel,doxorubicin,platinate,andcampthothecin) with poly glutamate and non biodegradable hydroxyl propyl methacrylamide.

Clinical success of nanoparticle-based diagnostics and therapeutics requires proper matching of Particle characteristics. The characteristics of nanoparticles are critically dependent upon the Materials used to prepare the nanoparticle. Nanoparticles can now be readily prepared from a wide range of inorganic and organic materials in arrange of sizes from two to several hundred Nanometers (nm) in diameter. Put in perspective, human cells are typically 10,000-20,000 nm in diameter. The plasma membrane of these cells is 6nm in thickness. In most cases, nanoparticles can be generated to have narrow and defined size ranges. Other chapters in this text will focus on the physical and chemical characteristics of nanoparticles made from various materials as well as methods for their production.

Although nanoparticles can be prepared from a wide variety of materials in organic salts, lipids, synthetic organic polymers, polymeric forms of aminoacids, nucleicacids, etc.), this chapter will primarily focus on those prepared from materials that would be considered sufficiently safe for repeated systemic administrations and/or would be perceived to have an acceptable safety profile that would warrant use in man. In general, it is desirable for nanoparticles to be either readily metabolized or sufficiently brokendown to produce only non-toxic metabolites that can be safely excreted. Indeed, tremendous advances have been made in Controlling the chemical nature, degradable characteristics, and dimensions of nanoparticles.

One of the biggest concerns regarding poorly metabolized nanoparticles is that of accumulation and the potential sequelae associated with such an outcome. In some cases where a limited number of exposures would occur, one could consider the use of materials that are not readily metabolized by thebody. In the case of certain cancer applications, it might be possible to use materials that otherwise would be considered to have an unacceptable safety signal following repeat dosing or that have the potential toaccumulate.

Methods of production and composition define nanoparticle characteristics; these charac-teristics define potential issues (andopportunities) related to biocompatibility, derivitization, and detection. Nanoparticles can be prepared from a singular subunit that is chemically coupled and organized in a defined (e.g.,dendrimers) or in a more random (e.g.,polylacticacid) manner.

Although these materials would not have a defined core, they can be impregnated with compatible materials and/or chemically modified at their surface. Materials such as glyconanoparticles would provide one approach where a distinct core with radiating ligands could be positioned using linkers. In such a case, the solidcore, used to anchor each linker used for the attachment of targeting ligands, could be used to deliver a therapeutic or diagnostic payload. Liposomes are an example of nanoshell structures that can be loaded internally as well as impregnated within the shell. Many types of nanomaterials fall in to one of these three general structural architectures.

Nanoparticles can also be prepared from biological materials that are found in the body but are not typically organized as nanoparticle-size polymers; complex mixtures of polysaccharides, poly-lysine,andpoly(D,L-lacticandglycolicacids;PLGA) have been prepared in a variety of sizes and in formats that allow ligand coupling with targeting moieties as well as diagnostic ortherapeutic agents. Synthetic organic polymers such as PLGA have been used to produce resorbable sutures, providing nanoparticles that will produce a sustained release of its contents. PLGA nanoparticles have been used to deliver wild-type p53 protein to cancercells. Such polymers have been studied for the development of nanoparticle delivery vehicles. Although such nanoparticles would be considered relatively safe because of their biocompatibility, particles prepared from these types of materials can initiate inflammatory response at sites of accumulation or deposit.

Nanotechnology for cancer theraphy

General schema for three types of nanoparticle structures.

(a)Nanoparticles can be formed from one type of material that can be impregnated with the therapeutic or lipophilic imaging reagents (opendiamonds) and modified with targeting ligands (crescents) positioned by chemical coupling through linkermoieties.

(b) Metal (orsimilar) cores (circles can be modified through a linker-targeting ligand system to generate another type of nanoparticle structure. In this case, it might be possible to use elaborated linkers as an environment compatible for incorporation of therapeutic or imaging reagents.

(c)Shell-type nanoparticles such as liposomes where an aqueous compartment is enclosed by a bilayer of phospholipids can also be used for the targeted delivery of hydrophilic therapeutic or imaging reagents (filled hexagons).

Nanoparticles can also be prepared from biological materials that are found in the body but are not typically organized as nanoparticle-size polymers; complex mixtures of polysaccharides, poly-lysine,andpoly(D,L-lacticandglycolicacids;PLGA) have been prepared in a variety of sizes and in formats that allow ligand coupling with targeting moieties as well as diagnostic ortherapeutic agents. Synthetic organic polymers such as PLGA have been used to produce resorbable sutures, providing nanoparticles that will produce a sustained release.

PLGA nano particles have been used to deliver wild-type p53 protein to cancercells. Such polymers have been studied for the development of nanoparticle delivery vehicles. Although such nanopar- ticles would be considered relatively safe because of thei rbiocompatibility, particles prepared from these types of materials can initiate inflammatory response at sites of accumulation or deposit.

A variety of inorganic materials can be used to generate nanoparticles for drug delivery that nanoparticles can might be applied to cancer and tumor targeting. Forexample, ironoxide (Fe O) be used to deliver anti-canceragents.Nanoparticles targeted to a cancer can be come hot enough in anapplied oscillating magnetic field to kill cells. Calcium phosphate precipitates can also be also made into nanoparticles.Although calcium phosphate precipitates can be metabolized over time and would be considered biocompatible, these materials can act as a potent adjuvant, potentially enhancing their application to target the delivery of cancer antigens. In this way,calcium phosphate nanoparticles are simialar to another inorganic salt precipitate, aluminumhydroxide Semiconductornanocrys- (alum),that is currently approved as an adjuvant for human vaccines.

Semi conductor nano crystals (quantumdots) are another example of inorganic nanoparticles. Quantum dots have exceptional characteristics for invivo imaging and diagnostic applications. However,some materials used to generate quantum dots such as CdSe can release toxic Cd²+ ions and lead to cell death when sufficient levels are reached. Therefore, some of the inorganic materials used to generate nanoparticles may have significant biocompatibility issues.

Because of their chemical and physical characteristics, nanoparticles exhibit inherent cellular targeting and uptake characteristics.Size and surface charge seem to be the two prominent characteristics that affect inherent nanoparticle targeting and cellular uptake. Because inherent targeting mechanisms may not provide the targeting or delivery characteristics desired, methods to modify nanoparticles with targeting agents can be critical. Although some nanoparticle materials are composed of materials with functional group useful for chemical coupling, others are not. Such nanoparticle systems must be either modified to allow chemical coupling or doped with reagents that can be used for this modification.

A number of coupling strategies have also been worked out that allow for efficient functionalization of nanomaterials through both reversible and irreversible chemistries. These modifications allow for the coupling of antibodies, receptor, ligands, and other potential targeting agents. Similar to the concerns associated with composition of the nanoparticle itself, any modification through chemical derivitization must also be considered with regard to generating materials with unacceptable toxicity or neutralization of the function of the nano-particle or its targeting element.

Nanoparticles have the advantage that they can be modified with multiple ligands to enhance their targeting selectivity and/or allow for simultaneous delivery of diagnostic and therapeutic agents. It is important to appreciate the relative size of components used to construct and deri-vitize nanoparticles. Forexample, a quantum dot may be only 10 nm in diameter. Targeting that Sized particle with an antibody might require the attachment of an IgG antibody that is roughly equal in size. By comparison, a fluorescent material that might be useful for localization of a targeted nanoparticles such as green fluorescent protein (GFP) is about 5 nm. Derivitization strategies for the construction of targeted nanoparticles must incorporate a consideration of potential steric conflicts for incorporation of targeting, detection, and therapeutic components.

Nanoparticles can be designed in a variety of ways to achieve targeted delivery.Some targeting strategies rely upon inherent properties of the particle, inparticular ,itscomposition ,size ,and surfaceproperties. Furthermore, the particle itself can either be the agent being delivered, or it can be prepared to carry a cargo for delivery.Cargo release from the nanoparticles can occur while the nanoparticle is still relatively intact or through its decomposition.

A number of methods have been described to integrate and retain cargo components within nanoparticles and these, in general, match to chemical or physical characteristics of the cargo with those of the material used to generate the nanostructure. For example ,positively charged cargo can be held within the nano-particle through interactions with an internal network such as a polyanionic polymer that resembles Alternately, organized complexes the organization of secretory granules synthesized by cells.

Alternatively organized complex akin to coacervates proposed to participate in cell structure evolution can be formed between cargo Therefore, for some cancer- targeting strategies, one should consider not only compatibility of the nanoparticle carrier with its cargo but also degradation events that migh taffect temporal aspects of particle stability and cargo release. Some nanoparticles can be designed or delivered in such a way as to produce a default targeting event; other nanoparticles must be decorated on their surface to produce a targeted structure.

Topical application of nanoparticle System at the target site may be all that is required for a successful outcome. Such as simple approach is not typically sufficient for effective targeting of many cancers. Successful targeting may require eduction of inherent targeting tendencies for the material(s) used to prepare the nanoparticle. Depending upon the physical and chemical nature of the nanoparticle and the mode of administration, there can also be complicating factors that affect the effectiveness of the targeting method. Inherent targeting and complicating factors associated with some nanoparticles used for a targeted delivery can impart safety issues that must also be considered. With such characteristics, it is easy to see why active targeting of nanoparticles to cancers can be both complicated by competing biological events as well as facilitated by these same properties.

The RES is composed of series of sentinel cells located in several highly perfused organs,including the liver and spleen.Nanoparticles can be rapidly cleared from the blood if they are recognized by RES cells in a non-selective fashion, typically before achievement of effective targeting. In some instances, this inherent targeting can provide a means to selectively delivery materials. In most cases, it is possible to modify the physical and chemical characteristics of materials.

Methods to avoid the RES will be nanoparticles to reduce their default uptake by the RES. .In general, these measures follow principles initially outlined in the development of stealth liposomes that provided a means of extending the circulating half-life of a nanoparticle. Although PEG molecules of various lengths coupled using are frequently used in this approach, heparin sulfate glycosaminoglycans (HSGs) have also been shown to provide a protective coating that reduces immunedetection. Interestingly, HSGs might be shed at tumors by tumor-associated heparanase activity.

Another inherent targeting aspect of nanoparticles relates to the nature of tumor-associated vasculature. Ingeneral ,nanoparticles smaller than 20nm have the ability to transit out of blood vessels. Solid tumors growrapidly; tumor-associated endothelial cells are continually bathed by a plethora of cancer cell- secreted growth factors. Inturn ,endothelial cells sprout new vessels to provide needed nutrients for the continued growth of th tumor.This cancercell- endothelial cell relationship, however, leads to the establishment of a poorly organized vasculature that, under the constant drive of growth factorstimulation ,fails to organize into a mature vascularbed.Therefore, tumor-associated vascular beds are poorly organized and more leaky that normalvasculature. Nanoparticles will inherently target to tumor exudates through leaky vasculature. This phenomenon referred to as the enhanced permeability and retention(EPR) effect.

Finally, peculiar surface properties of certain nanomaterial might affect their inherent interactions that could act to detract from at targeted delivery strategy.For example, some poly- anionic dendrimers can be taken up by cells and act within those cells to interfere with replication of human immunodeficiency virus(HIV) that is considered to be the causative agent of AIDS. Although, from such studies ,it is unclear if these dendrimers interact with the hostcell or the pathogen to block their interaction; such a finding points to the potential for nanoparticles to interact with structures that might affect their cellular properties or cellfunction. In some cases, a nanoparticle with inherent capacity to interact with a cell or tissue might provide an added advantage of using that material for a specific indication.In othercases, such an inherent capacity to bind to recognize cell or tissue components might highlight potential distractive aspects of that material for certain indications.

By their eponymous descriptor ,nanoparticles have physical dimensions in the nanometer size scale similar to viruses and other materials that are either recognized by the body as pathogens or are elements associated with an infective event. Toll-like receptors (TLR ) present on monocytes, leukocytes, and dendritic cells play a critical role in innate immunity with the capacity to recognize TLR proteins that are present on the surface of cells in the lung, spleen, prostate, liver, and kidney. Because the patterned surfaces of nanoparticles can look like pathogen components recognized by TLR proteins such as DNA, RNA ,and repeating proteins like flagellin, it is possible that a number of cell types might non- selectively interact with some nanoparticles.

If such an interaction occurs, there are several potential outcomes that might produce complicating aspects for nanoparticle targeting to cancers. Nanoparticle materials might be immediately recognized and cleared by cells of the innate immune system, limiting the usefulness of even their initial application .Alternately, only a fraction of the applied nanoparticles might engage TLR that would not significantly affect the effectiveness of the administration. Recognition of even a small fraction of the administered nanoparticles, however ,might lead to immune events that diminish the effectiveness of subsequent administrations. Nanoparticles that the naïve body initially tolerates may become a focus of immune responses upon repeated exposure.

Although generalization can be made for a particular targeted nanoparticle delivery system, specific issues will arise for each type of payload it contains and for each indication.It will be important to balance potential safety concerns for using nanoparticles with their potential benefits for reducing a safety concern that occurs without their use for comparable (or even improved) efficacy. Nanoparticles can tremendously reduce toxicity by sequestering cytotoxic materials from Polymeric nanoparticles have been loaded with tamoxifen for targeted delivery to breast cancer cells to improve the efficacy to safety quotient relative to direct administration of this cytotoxic agent. Coupling of doxorubicin- loaded liposomes to antibodies or antibody fragments that can enhance targeting to cancer cells appears quite promising as a means of further improving the efficacy to toxicity profile for this PEGylated liposomal doxorubicin has improved tolerability with similar efficacy compared to free drug.

PAMAM dendrimers have also been used to increase the effectiveness of a radioimmuno-therapy approach by increasing the specific accumulation of radioactive atoms at a tumor site as well as improving selectivity of biodistribution.

Successful targeting of nanoparticles to cancers or tumors may involve overcoming multiple biological, physiological ,and physical barriers site of initial application can be critical. For example, nanoparticles administered into the gut or lung would initially confront epithelial barriers. Metabolic events or cellular responses at an injection site represent another initial barrier to targeted nanoparticle delivery.

Once nanoparticles have entered the body, their size ,shape ,or surface characteristics can initiate events that present a second barrier to targeted delivery is direction of the material away from its targeted site through undesired interactions. Access and /or enriched distribution to specific organs or regions of the body may be critical for successful nanoparticle targeting. Finally ,once nanoparticles have reached a targeted site, metabolic or physical aspects of the cancer cell or tumor might limit their effectiveness. Here, the intracellular uptake and fate may dictate the potential success of each nanoparticle approach. Events at each of these barriers act in a cumulative fashion to limit the success of any nanoparticle-based targeting strategy. Obviously, the overall fate of nanoparticles might be improved by using materials that are not affected by these barriers or by modification of nanoparticles in ways that can neutralize these barrier issues. Matching the size and composition profile of a nanoparticle delivery system with the targeting strategy allows for an optimized approach that takes advantage of default targeting events as much as possible.

Several extracellular barriers exist for the administration and targeted delivery of nanoparticles. Initial entry into the body represent on obvious barrier. This is not an issue for situations where the cancer cell or tumor targeted is readily accessible by a topical application. Such a situation however, is rather rare. Entry into the body across mucosal surfaces such as those in the gut or lung is typically very inefficient. Even viral particles are not very successful at this approach with most relying upon infecting cells of the barrier from the apical exposure by only a few viral particles that can replicate inside the cells to allow the basolateral (systemic) release of large numbers of progeny. Viral particle entry at apical surfaces of epithelial cells is decreased by physical barriers such as secreted mucus as well as proteases and other enzymatic barriers. Extracellular (acellular) matrix environments that viruses might encounter after systemic infection could similarly act to diminish cellular targeting and entry.

Man-made nanoparticle delivery systems are likely to be impeded by these same physical and biological barriers at epithelial surfaces and within the body. Reduced surface exposure of highly charged or protruding structures is commonly used by viruses to minimize the impact of these extracellular barriers on viral infectivity. Similar considerations may facilitate optimization of nanoparticle delivery strategies.

There are several common methods for administering materials to the body :injection or application to an epithelial surface (skin, intestine, lung, etc.) of the body. Nanoparticles can be absorbed .Although nanoparticles can be taken up through appendages into the skin after topical application. Micro needles can be used to dramatically increase the efficiency of their uptake into and across skin. The intravitreous injection of nanoparticles results in transretinal movement with a preferential localization in retinal pigment epithelial cells, allowing for a sustained delivery strategy to the inner eye.

Nanoparticles can be absorbed from the lumen of the gut, but this absorption is inefficient. Number of factors have been examined related to regulation of nanoparticle uptake from the gut Nanometer-sized liposomes enter into the intestinal mucosa better than larger, multi-lamellar liposomes, and this uptake can be improved by coating with a muco adhesive polymer such as chitosan. A large number of studies have been performed to assess nanoparticle absorption following inhalation exposure, and concerns over the safety of such an approach for drug delivery have been raised. Nanoparticles deposited in the airways appear to be taken up through transcytosis pathways that allow the passage of these materials across epithelial and endothelial cells to reach the blood and lymphatics. Surface properties of nanoparticles greatly affect the capacity of nanoparticles to be taken into cells through the process of endocytosis and uptake following pulmonary deposition. As part of the respiratory tree, intranasal administration of nanoparticles can potentially provide a route into the brain.

Targeting to some cancers may require overcoming additional hurdles beyond interaction with specific cancer cell or tumor components. Some tumors are located in difficult -to-reach sites such as the brain and testes.

Accessing these sites from the systemic vasculature requires that nanoparticle materials must first avoid systemic clearance by the RES and have the capacity to move across either the blood -brain or blood - testes barrier. Where as some types of nanoparticles can keep materials out of compartments of the body such as the brain.

Nanoparticles provide a range of new oppurtunities to the trgeting of currently approved diagnostic and therapeutic agents to cancers. Improvements in targeting can lead not only to increased efficacy of these agents but also increased signal to noise ratios for diagnostics and better efficacy to toxicity ratios for therapeutics.

POLYHYDROXYBUTYRATES (PHB) is a polyhydroxyalkanoate (PHA), a polymer belonging to the polyesters class that was first isolated and characterized in 1925 by French microbiologist Maurice Lemoigne. PHB is produced by micro-organisms (like Alcaligenes eutrophus or Bacillus megaterium) apparently in response to conditions of physiological stress. The polymer is primarily a product of carbon assimilation (from glucose or starch) and is employed by micro-organisms as a form of energy storage molecule to be metabolized when other common energy sources are not available. Microbial biosynthesis of PHB starts with the condensation of two molecules of acetyl-CoA to give acetoacetyl-CoA which is subsequently reduced to hydroxybutyryl-CoA. This latter compound is then used as a monomer to polymerize PHB.[1]

Chemical structures of P3HB, PHV and their copolymer PHBV

The poly-3-hydroxybutyrate (P3HB) form of PHB is probably the most common type of polyhydroxyalkanoate, but many other polymers of this class are produced by a variety of organisms: these include poly-4-hydroxybutyrate (P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO) and their copolymers.

RIFAMPICIN

Rifampicin (INN) or rifampin (USAN) is a bactericidal antibiotic drug of the rifamycin group and a semisynthetic compound derived from Amycolatopsis rifamycinica .In 1957, a sample of soil obtained from a pine wood on the French Riviera was brought for analysis to the Lepetit Pharmaceuticals research lab in Milan, Italy. There, a research group headed by Prof. Piero Sensi (1920-) discovered a new bacterium. After two years they obtain more stable semi-synthetic products, in 1959 a new molecule with high efficacy and good tolerability was produced and was named "Rifampicin".

Rifampicin inhibits DNA-dependent RNA polymerase in bacterial cells by binding its beta-subunit, therefore preventing transcription to RNA and subsequent translation to proteins. Its lipophilic nature makes it a good candidate to treat the meningitis form of tuberculosis, which requires distribution to the central nervous system and penetration through the blood-brain barrier.

Fig: 1

Fig :1 - Rifampicin

Rifampicin acts directly on messenger RNA synthesis. It inhibits only prokaryotic DNA-primed RNA polymerase, especially those that are Gram-stain-positive and Mycobacterium tuberculosis. Evidence shows that in vitro DNA treated with concentrations 5000 times higher than normal dosage remained unaffected; in vivo eukaryotic specimens' RNA and DNA polymerases suffered few problems as well.[5][9] Much of this acid-fast positive bacteria's membrane is mycolic acid complexed with peptidoglycan, which allows easy movement of the drug into the cell. Rifampicin interacts with the β subunit of RNA polymerase when it is in an α2β trimer. This halts mRNA transcription, therefore preventing translation of polypeptides.[5] It should be made clear, however, that it cannot stop the elongation of mRNA once binding to the template-strand of DNA has been initiated.[10] The Rifampin-RNA polymerase complex is extremely stable and yet experiments have shown that this is not due to any form of covalent linkage.

It is hypothesized that hydrogen bonds and π-π bond interactions between naphthoquinone and the aromatic amino acids are the major stabilizers, though this requires the oxidation of naphthohydroquinone which is found most commonly in Rifampin.

It is this last hypothesis that explains the explosion of multi-drug-resistant bacteria: mutations in the rpoB gene that replace phenylalanine, tryptophan, and tyrosine with non-aromatic amino acids result in poor bonding between Rifampin and the RNA polymerase.[5]

Rifampin-resistant bacteria produce RNA Polymerases with subtly different β subunit structures which are not readily inhibited by the drug.[11] In molecular biology research, plasmids containing rifampicin-resistant genes are often used for colony screening. Many plasmids containing these resistant genes are commercially available to researchers.

Literature survey :

Nanomaterials are used for targeted delivery, localization of drug. The high local concentration of drugs or image contrast agents at the target sites will improve system performance and also reduce systemic dosing. We discussed about the targeted nanodelivery system, invivo performance, physio chemical properties that affect localization at the target site and encapsulation of therapeutic drugs into these systems.

(Targeted nano delivery of drugs and diagnostics MargaretA.Phillips , Martin L. Gran , Nicholas A. Peppas )

Significant progress has been made in the development of new agents against cancer and new delivery technologies. Proteomics and genomics continue to uncover molecular signatures that are unique to cancer. Yet, the major challenge remains in targeting and selectively killing cancer cells while affecting as few healthy cells as possible. Nanometer-sized particles have novel optical, electronic, and structural properties that are not available from either individual molecules or bulk solids. When linked with tumor-targeting moieties,such as tumor-specific ligands or monoclonal antibodies, these nanoparticles can be used to target cancer-specific receptors, tumor antigens (biomarkers), and tumor vasculatures with high affinity and precision.

(Targeted cancer nanotherapy Gloria J. Kim and Shuming Nie)

Targeted delivery of drug molecules to organs or special sites is one of the most challenging research areas in pharmaceutical sciences.By developing colloidal delivery systems such as liposomes, micelles and nanoparticles a new frontier was opened for improving drug delivery. Nanoparticles with their special characteristics such as smallparticlesize,large surface area and the capability of changing their surface properties. Have numerous advantages compared with other delivery systems.Targeted nanoparticle delivery to the lungs is an emerging area of interest.

( Targeted delivery of nanoparticles for the treatment of lungdiseases ShirzadAzarmi , Wilson H. Roa , Raimar Löbenberg)

Nanotechnology applications in medicine, termed as nanomedicine, have introduced a number of nanoparticles of variable chemistry and architecture for cancer imaging and treatment. Nanotechnology involves engineering multifunctional devices with dimensions at the nanoscale, similar dimensions as those of large biological vesicles or molecules in our body. These devices typically have features just tens to hundred nanometers across and they can carry one or two detection signals and/or therapeuticcargo(s). One unique class of nanoparticles is designed to do both, providing this way the theragnostic nanopar-ticles (therapyanddiagnosis). Being inspired by physiologically existing nanomachines, nanoparticles are designed to safely reach their target and specifically release their cargo at the site of the disease, this way increasing the drug's tissue bioavailability. Nanoparticles have the advantage of targeting cancer by simply being accumulated and entrapped in tumours(passivetargeting).The phenomenon is called the enhanced permeation and retention effect, caused by leaky angiogenetic vessels and poor lymphatic drainage and has been used to explain why macromolecules and nanoparticles are found at higher ratios in tumours compared to normal tissues. Although accumulation in tumours is observed cell uptake and intracellular drug release have been questioned. Polyethyleneglycol (PEG) is used to protect thenanoparticlesfromtheReticulo-EndothelialSystem RES), however, it prevents cell uptake and the required intracellular drug release.Grafting biorecognition molecules (ligands) onto the nanoparticles refers to active targeting and aims to increase specific celluptake. Nanoparticles bearing these ligands are recognized by cellsurface receptors and this leads to receptor-mediated endocytosis. Several materials are suggested for the design of nanoparticles . Polymers, linear and dendrimers, are associated with the drug in a covalent or non-covalent way and have been used with or without a targeting ligand. Stealth liposomes are suggested to carry the drug in the aqueous core, and they are usually decorated by recognition molecules, being widely studied and applied.Inorganic nanoparticles such as gold and iron oxide are usually coupled to the drug, PEG and the targeting ligand.It appears that the PEG coating and ligand decoration are common constituents in most types of nanoparticles for cancer.

(Targeting nanoparticles to cancer M.Wang , M. Thanou )

Targeted drug delivery aims to increase the therapeutic index by making more drug molecules available at the diseased sites while reducing systemic drug exposure. These systems use water-soluble polymers as the drug carriers. The preclinical pharmacology and recent data in clinical trials with poly(synthetic L-glutamicacid)-paclitaxel(PG-TXL) arediscussed. This is followed by a summary of a variety of polymeric conjugates with chemotherapeutic agents. Results from early clinical trials of these polymer-drug conjugates have demonstrated several advantages over the corresponding parentdrugs, including fewer side effects, enhanced therapeutic efficacy, ease of drug administration, and improved patient compliance. Collectively, these data warrant further clinical development of polymer-drug conjugates as a new class of anticancer agents.

(Polymer-drugconjugates: Recent development in clinical oncology ChunLi , Sidney Wallace)

Nanomedicine is the application of nanotechnology to medicine. Consider several biodegradable polymeric nanomedicines that are between 1and 100 nm in size, and discuss the impact of this technology on efficacy, pharmacokinetics, toxicity and targeting.The degree of toxicity of polymeric nanomedicines is strongly influenced by the biological conditions of the local environment, which influence the rate of degradation or release of polymeric nanomedicines. The dissemination of polymeric nanomedicines invivo depends on the capillary network, which can provide differential access to normal and tumor cells. The accumulation of nanomedicines in the microlymphatics depends upon retention time in the blood and extracellular compartments, as well as the type of capillary endothelium surrounding specific tissues.Finally, the toxicity or efficacy of intact nanomedicines is also dependent upon tissue type, i.e., non-endocrine or endocrine tissue, spleen, or lymphatics, as well as tumortype.

(Factors affecting toxicity and efficacy of polymeric nanomedicines EikiIgarashi )

For the treatment of periodontaldiseases, design of a controlled release system seemed very appropriate for an effective, long term result. In this study a novel, biodegradable microbial polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV of various valerate contents containing a well established antibiotic, tetracycline, known to be effective against many of the periodontal disease related microorganisms, was used in the construction of a controlled release system.Tetracycline was loaded in the PHBV microspheres and microcapsules both in its acidic (TC) and in neutral form (TCN). Microcapsules of PHBV were prepared under different conditions using w/o/w double emulsion and their properties such as encapsulation efficiency, loading, release characteristics, and morphological properties were investigated. It was found that concentration of emulsifierspolyvinylalcohol(PVA) and gelatin (varied between 0-4%) influenced the encapsulation efficiency appreciably. In order to increase encapsulationefficiency (from the obtained range of 18.1-30.1%) and slowdown the release of the highly soluble tetracycline. HCl, it was neutralized with NaOH. Encapsulation efficiency of neutralized tetracycline was much higher(51.9-65.3%) due to the insoluble form of the drug used during encapsulation.

(Antibiotic release from biodegradable PHBV microparticles DilekSendil, IhsanGursel, DonaldL.Wise, Vasif Hasirci )

Aim:

Is to synthesize and characterize the PHBV microspheres loaded with rifampicin for the treatment of lung cancer.

Objective:

To synthesize the PHBV microspheres

Encapsulation of drug Rifampicin with the PHBV polymer

To study the drug relase kinetics

MATERIALS AND METHODS

Materials:

PHBV (Good fellow, USA), Rifampicin, Polyvinyl alcohol (sigma-aldrich)

Microsphere preparation :

Poly(3-hydroxy butyrate -co-3-hydroxy valerate) (PHBV) was dissolved in dichloromethane(DCM) and the polymer solution (5%) was added to 1% poly(vinyl alcohol) and stirred using the mechanical stirrer until the solvent was evaporated.

Drug loaded microsphere:

Drug used was rifampicin and rifampicin loaded PHBV microspheres were formed by solvent evaporation process involving the formation of single emulsion method (w/o).In this first PHBV was dissolved in DCM and drug is added to DCM dissolved PHBV.Then we add PVA to water and kept stirred in mechanical stirrer. Then we add PHBV and drug dissolved in DCM to 200 ml of 1% PVA solution under constant stirring using the mechanical stirrer.The drug along with the PHBV was stirred for 4 hours to allow complete solvent evaporation.The microspheres formed were collected by filtering it with ordinary filter paper. Then microspheres were allowed to dry overnight to obtain dried microspheres.

Morphology characterization:

The surface morphology of microspheres and scaffold was investigated by using scanning electron microscope (SEM). The freeze-dried microspheres were mounted onto metal stubs using double-sided adhesive tape, sputter-coated with a thin platinum layer using an Auto-sputter fine coater (JFC 1600, JEOL, Japan) and directly analyzed by cold field emission SEM (JEOL, JSM-6701F, Japan).

Particle size analysis:

The particle size of the PHBV were determined using laser diffraction method (Microtrac Blue wave, Japan) at room temperature. Few mL of the sample was added with the water and introduced into the particle size analyzer at 50% flow rate to measure the mean size and size distribution of PHBV microspheres.

Drug loading efficiency:

The microspheres were dissolved in dichloromethane (2 mL) under shaking for 5 minutes.Then 2 mL of phosphate buffer saline (PBS, pH 7.4) was added and medium was agitated for 2 minutes.It was then centrifuged for 10 min at 6000 rpm and the aqueous phase was transferred to 10 ml test tube.The aqueous phase contains the free drug and the concentration of free drug was determined spectrometrically at 450 nm using uv spectrometry (Lambda 25, Perkin Elmer, USA).From the data obtained, the percentage of rifampicin encapsulated in microsphere was calculated.The percentage of encapsulation efficiency was determined as follows :

% Encapsulation efficiency = (Total amount of drug loaded-Amount of free drug / total amount of drug loaded) * 100

Drug release kinetics :

Drug loaded microspheres were placed in the dialysis bag and 3 mL of PBS(pH 7.4) was added to it.Then dialysis bag was placed in the beaker containing PBS solution and dialysis bag was completely immersed in the solution of PBS.After half an hour 3 mL of PBS containing dissolved drug was collected in the eppendorf tube and the 3 ml of fresh PBS was added to the beaker.Then PBS was collected at regular interval of one hour until the entire loaded drug was released into PBS medium.The samples collected in eppendorf tubes were measured using UV visible spectroscopy(Lambda 25, Perkin Elmer, USA) and values obtained were plotted in graph. The invitro drug release were done for 3 trials.

RESULTS AND DISCUSSION

The morphology of samples prepared at different stirring speeds were observed under the Scanning Electron Microscope (SEM) .The unencapsulated microspheres in first trial (200 rpm) showed a smooth morphology. Pores formed in subsequent trials with higher rpm. This is due to higher rate of evaporation of the solvent (DCM) from the system. However the size as well as the size distribution of the microspheres varied in each trial. The particle size analysis was carried out using laser diffraction for each trial using particle size analyser (PSA).

a b

Micrograph of 5% PHBV microspheres (solvent: DCM) at 200 rpm

Micrograph of 5% PHBV microspheres (solvent: DCM) at 300 rpm

c d

Micrograph of PHBV microspheres (solvent :DCM) at 400 rpm

Micrograph of PHBV microspheres (solvent :DCM) at 500 rpm

e f

Micrograph of PHBV microspheres (solvent: DCM) at 700 rpm

f. Micrograph of PHBV microspheres (solvent: DCM) at 800 rpm

Particle size analysis :

PHBV microsphere particle size analysis

PSA by laser diffraction method of PHBV microspheres formed at 600 and 800 rpm were obtained. We get a homogeneous distribution for both the type of PHBV microspheres.

PHBV microsphere loaded with Rifampicin Particle size analysis

PSA by laser diffraction method of PHBV microspheres loaded with rifampicin was obtained at 600 and 800 rpm. We get a homogeneous distribution for both the type of PHBV microspheres loaded with rifampicin.

PHBV microspheres loaded with rifampicin

Drug release profile :

Rifampicin drug release

Drug release follows the first order kinetics and the drug is uniformly spread inside the surface of PHBV microspheres. So matrix type of drug release occurs. So we got almost a linear curve and there is no burst release.Drug was released for 20 hours.

CONCLUSION :

PHBV microspheres loaded with rifampicin was obtained in the range of 100 -200 µm. Drug release studies were carried out and we got the almost linear drug release and matrix drug delivery system is followed.

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