Antimicrobial Activity of Clarithromycin Loaded Mesoporous Silica Nanoparticle

1. INTRODUCTION

­­­­­­1.1 Mesoporous silica nanoparticle

The era of nanotechnology has revolutionized the drug delivery and targeting process and changed the landscape of the pharmaceutical industry. Nanoparticles have dimension below 0.1 μm or 100 nm especially in the drug delivery. “The drug is dissolved, entrapped, encapsulated or attached to a nanoparticle matrix. Nanoparticles size range also affects the bioavailability and bio-distribution of particles, and hence it is useful as a drug carrier. Hydrophobic core is beneficial for drug loading while hydrophilic surface blocks opsonization and allow easier movement in the system.” Numerous Nano devices have been reported like carbon nanotubes, quantum dots, and polymeric micelles, etc., in the field of nanotechnology. In the present scenario, mesoporous nanoparticles are emerging for their well-known drug deliver and targeting purposes incorporated within amorphous silica matrix.¹ “Mesoporous silica nanoparticles are solid materials, which contain hundreds of empty channels (mesoporous) arranged in a 2D network of honeycomb-like porous structure in contrast to the low biocompatibility of other amorphous silica materials.” Mesoporous silica nanoparticles exhibit superior biocompatibility at concentrations adequate for pharmacological applications.

Figure 1.1: “Mesoporous Silica Nanocarriers as Nanomedical Multifunctional Nanoplatforms.

These silica-based nanoparticles also offer several unique and advantageous structural properties, such as high surface area (>700 m² g − 1), pore volume ( > 1 cm³ g − 1 ), stable mesostructure, tunable pore diameter (2–10 nm),” two functional surfaces (exterior particleand interior pore faces), and modify able morphology (controllable particle shape and size). ²

Among many Nano sized materials, a class of nanomaterials named mesoporous silica nanoparticles stands out. “Mesoporous term describe pore volume between 2 to 50 nm,beyond 50 nm it will became macroporous material.Mesoporous silica nanoparticles have tunable nano scale sizes, different shapes ranging from spheres to rods, uniform cylindrical mesopores, high surface areas, and easily functionalizable surfaces. Owing to these interesting structural features, the potential applications of Mesoporous silica nanoparticles as effective delivery vehicles for pharmaceuticals and bioactive molecules (e.g., nucleotides) to desired intracellular sites or as host materials for bioimaging”, biocatalytic, and biosensinghave been widely recognized and biosensing agents have been.³Compared to traditional organic DDSs, such as liposomes or emulsions, MSNs exhibit the typical characteristics of inorganic nano-biomaterials, such as high thermal/chemical stability, tuneable biocompatibility/degradability and resistance to corrosion under extreme conditions. “The abundant surface silane chemistry facilitates the easy chemical modifications of MSNs. The traditional liposome-based DDSs suffer from the low stability in physiological environment, which may result in uncontrollable drug releases in an explosive manner.” Comparatively, MSNs can release the loaded cargos in a sustained manner partially due to their high stability. ⁴

The msn spore size can be designed slightly larger than the dimension of the drug molecule and thus to provide sustained release for several hours. “In addition, Mesoporous silicananoparticles present high drug loading capacities ranging from 10to 34% or even to 60% in rare cases. The presence of surface silanol groups facilitates msn’s functionalization by various groups (hydroxyl, amine, thiol, and carboxyl) which can then conjugate with fluorophores and target ligands for optical imaging of tumor cells in vitro.⁵

The unique topology provides mesoporous silica nanoparticles with three distinct domains that can be independently functionalized: the silica framework, the nanochannels pores, and the nanoparticle’s outermost surface.” As such, MSNs are especially well-suited to the task of incorporating the essential capabilities of a theranostic platform in a single particle, with separate domains for

(1) The contrast agent that enables traceable imaging of theranostic target.

(2) The drug payload for therapeutic intervention.

(3) The bimolecular ligand for highly targeted delivery.⁶

“Mesoporous silica nanoparticles possess several attractive features such as large surface areas and porous interiors that can be used to store various molecules.⁷A procedure for producing mesoporous silica was patented around 1970.^8”

Mesoporous silica nanoparticles were independently synthesized in 1990 by researchers in Japan.⁹ Initially, MSNs were discovered by Mobil scientists designated as mcm–41 or mcm–48 and used as molecular sieves. However, for first time mesoporous silica nanoparticle were used as drug delivery system by Valet–Regi to encapsulate Ibuprofen in mcm–41 of various pore sizes. In several occasions MSNs have been reported to control the release profile orto enhance the solubility of various active substances.⁵

1.2 Approaches for development of msn.

There is 3 approaches are available for controlling the pore size of msn, which are following

(a) Growth–quench approach

(b) Confinement approach

(c) Separation of nucleation and growth ⁶

(a)Growth–quench approach

A dilution and pH change method to quench the silica condensation reaction to obtainsub-100 nm mesoporous silica nanoparticles. “Using different “time-delay between dilution and neutralization steps, the particle size of the materials could vary from 23 to 100 nm. Other reaction-slowing agents have since been used, such as triethanolamine and alcohol co-solvents for their silicon chelatingability. However, due to the poor condensation of silica,” the resulting Mesoporous silica nanoparticles materials from pH quench are often less ordered” and less stable in solution. Moreover in the dilution quench approach.

(b)Confinement approach

If synthesis of mesoporous silica nanoparticles is done in a confined media, the particle size can be limited. An aerosol-assisted self-assembly of mesoporous spherical nanoparticles has been developed by Brinkerand co-workers. The method relies on evaporation-induced interfacial selfassembly confined to spherical aerosol “droplets. However, the method is not widely adopted because of the need of equipment for aerosol production.

(C)Separation of nucleation and growth

Mou and co-workers developed a method of synthesizing mono-disperse msn’s by separating the

“Nuclei formation and particle growth into two steps, One is nucleation and second one is growth. In the first step the full amount of surfactant (CTAB) and a small amount of TEOS are mixed to Form a clear solution of micelle/silicate clusters containing nuclei. Then, a larger amount of TEOS is added to start the growth process without finally,” with the growth process accelerating, the materials are exhausted resulting in a uniform finite size.

1.4 TYPE OF MESOPOROUS SILICA NANOPARTICLE

There is 3 type of mesoporous silica particle,¹

1. MCM-41
2. SBA-15
3. MCM-48

a) MCM-41

“MCM-41 can be synthesized as a highly ordered mesoporous material having a pore size ranging from about 2 to 8 nm; the pore system being uni dimensional and highly regular for each particular preparation. Synthesis of microsized and “monodispersed mcm-41 silica spheres is relatively easy to accomplish.MCM-41 type silica has been proposed as a convenient material for both drug and gene delivery.^10”The mcm-41 molecular sieve, which is a member of the m-41s family, exhibits a hexagonal arrangement of uniform cylindrical pores and is especially suitable as a model adsorbent.”¹¹The original and most common experimental pathway for mcm-41 synthesis starts from a solution of cationic surfactant molecules (most often cetyltrimethylammonium bromide or CTAB) in water to which a silica source is added (most often tetraethylorthosilicate or TEOS) under high-pH conditions¹²

b) SBA-15

“SBA-15 is a mesoporous silica sieve based on uniform hexagonal pores with a narrow pore size distribution and a tunable pore diameter of between 5 and 15 nm .The thickness of the framework walls is about 3.1 to 6.4 nm, which gives the material a higher hydrothermal and mechanical stability than, for instance,” MCM-41. SBA-15 is synthesized in a cooperative self-assembly process under acidic conditions using the triblock copolymer Pluronic 123 (EO20PO70EO20) as template and tetraethoxysilane (TEOS) as the silica source. ¹³

c) MCM-48

Siliceous MCM-48 materials can be synthesized from gels with a wide range of surfactant/silicon (Surf/Si) ratios. At a Surf/Si ratio of 0.12 MCM-48 and Mn-MCM-48 are synthesized in 3 days but various intermediate mesophases are observed dependent on the reaction temperature. ¹⁴

1.5 Synthesis process of msn

The synthesis of MSNs is achieved via a process called supramolecular self-assembly (also called soft-templating), which involves two major steps: “(1) the hydrolysis and condensation of different silica precursors such as tetraethoxysilane in the presence of ordered assemblies of surfactant micelle templates and (2) the removal of the surfactant templates by calcination or solvent extraction to generate the ordered mesoporous silica structures.Two mechanisms have been generally proposed to describe the synthesis of mesoporou”s silica nanoparticles The first one involves liquid crystal templating that proceeds through the following five steps:

(1) Formation of surfactant micelles in solutions

(2) Organization of the surfactant micelles into cylindrical micelles.

(3) Stacking of the cylindrical micelles in to a regular array of micelle liquid crystals.

(4) Adsorption of anionic silicates onto the positively charged surfaces of the micelle liquid     crystals.

(5) Removal of the surfactant micelle templates to produce the mesoporous silica structures.The    second and alternative pathway involves a cooperative assembly, which combines steps into a single concerted process that leads to a regular array of surfactant silica assemblies.

Figure1. 2 Schematic illustrations for the synthesis and selective functionalization of MSNs

In the synthesis of the hexagonal mcm-41 and cubic mcm-48, usually a quarternary ammonium surfactant as an ionic template, such as cetyltrimethylammonium bromide(CTAB), was used as a template to make the pore size around 2 to 10 nm. Charge matching has been recognized as the most critical factor in governing the final phases and stability. Liquid-crystal templating (LCT) mechanism was proposed by Beckto explain the formation mechanism of mcm-41.¹⁵

Figure 1.3 Possible mechanistic pathways for the formation of MCM-41: (1) liquidcrystal phase initiated and (2) silicate anion initiated.

1.6 Type of silica precursor used in mesoporous silica nanoparticle.¹

There is 3type of silica precursor are used which are following-

1. Organically modifi ed precursors
2. Glycerol-derived polyol-based silane precursors
3. Sodium metasilicate

a) Organically modified precursors

They are prevented hydrolysis because an organic group attached directly to a silicon atom, which does not need oxygen bridge. “It is conceded that organo-silica nanoparticles consist of better properties including large surface area, less condensed silioxane structure, and low density. The limited accessibility and high cost of organic template lead to its restricted use in practical applications. Commonly used silica precursors are glycerol-derived polyol-based silanes, orthosilicic acid, sodium metasilicate, tetraethyl orthosilicate (TEOS) or tetramethoxysilane (TMOS), and tetrakis (2-hydroxyethyl) orthosilicate. Tetraethyl orthosilicate or TMOS was commonly used in MSNs synthesis.” However, their poor water solubility requires additional organic solvent and alcohol and needs extreme conditions of pH and high temperature, which restricts their use. Tetrakis (2-hydroxyethyl) orthosilicate had been investigated to address the problems associated with TEOS and TMOS. “It is now used in many studies as MSNs precursor because it is more biocompatible with biopolymers, more water soluble. Than TEOS and TMOS, and can process jellification at ambient temperature with a catalyst”

b) Glycerol-derived polyol-based silane precursors

They are not pH dependent but very sensitive to the ionic strength of the sol. This can form optically clear monolithic MSNs. The residuals can be either removed or retained, therefore, the shrinkage during long-term storage can be minimized. Orthosilicic acid was used as a silica precursor in the past but due to the extensive time consumption and requirement of freshly prepared acid, so it is not widely used anymore now a day.

c) Sodium metasilicate

It is another precursor to sol-gel-derived silica. Formation of sodium chloride was investigated, which can cause a problem if a significant amount is generated. Later researches suggested that removing of this salt formulation by dialysis process, but it is a time and cost consuming procedure. Hence, alkoxides and pure alkoxysilanes are currently widely used.

1.7 Functionalization strategies of mesoporous silica nanoparticles

Mesoporous silicates usually have very high surface and their surfaces are covered by silanol groups, which makes the functionalization of the pore surface of the mesoporous materials adjustable. Additionally, “the surface functionalization of mesoporous silicates could change the chemical and physical properties of these materials dramatically.There are two major ways to functionalize the surface of mesoporous silicates by organic functional groups, named as postsynthesis grafting and cocondensation.” Each of these two functionalization methods has certain advantages, which will be described below.

a) Grafting Methods

b) Co-condensation Methods

(a) Grafting method

Grafting is a post-synthesis method to modify a pre-fabricated inorganic mesoporous material surface by attachment of functional groups to the surface of material, usually after surfactant removal . In the process of grafting mesoporous silicates, the surface silanol groups (Si-OH), which can be present in high concentration, act as convenientanchoring points for organic functionalization.

Figure1.4. Functionalization of mesoporous silicates by grafting

Surface functionalization with organic groups by grafting is most commonly carried out by silylation, which is accomplished by one of the three procedures,

Silylation occurs on free (≡Si-OH) and germinal silanol (=Si(OH)2) groups, but hydrogen bonded silanol groups are less accessible to modification because they form hydrophilic networks among themselves. The original structure of the mesoporous support is generally maintained after grafting.

(b) Co-condensation Methods

Co-condensation method is another strategy to functionalize mesoporous silicates surface by solgel chemistry between tetraalkoxysilane and one or more organoalkoxysilanes with Si-C bonds. Compared with the post-grafting method in which the distribution of functional groups often tends to be inhomogeneous, “the co-condensation is able to give homogeneously distributed organic groups on the entire inner pore surfaces and no pore-blockage or shrinkage problems have been reported. Even though bulky organoalkoxysilane precursors often perturb the original textural properties of the systems, some new methods already developed to decorate the surface wall efficiently while maintaining the mesoporous structure.” Another advantage of co condensation over post-synthesis grafting is to control the particle morphology of final mesoporous silicate very easily,which is closely related to the biomineralization process in nature, for example the complex of diatoms.¹⁵

Figure 1.5 Advantage and disadvantage between grafting and co-condensation method.

1.8 Inter cellular uptake of msn

For intracellular drug and gene delivery applications, this limitation has led to extensive research efforts on designing materials with precise control of the particle size. “Furthermore, it is important for the drug carrier to have proper surface properties that can have favourable interactions with the drug molecules to achieve high loading.” Cellular uptakes of molecules are often facilitated by the specific binding between these species and membrane-bound receptors (e.g. LDL or transferrin receptors). In contrast, materials for which cells do lack receptors can still be uptaken by constitutive “adsorptive” endocytosis or by fluid phase pinocytosis. Silicamparticles are known to have a great affinity for the head-groups of a variety of phospholipids Therefore, the high affinity for adsorbing on cell surfaces that eventually leads to endocytosis is not surprising.¹⁶

1.9 Biocompatibility of msns

1.9.1Interaction with cells and cytotoxicity

MSNs can easily be internalized into most normal and cancer cells without apparent deleterious effects on cellular growth, proliferation and differentiation, although the proliferation and cycle progression of msn-treated a375 human malignant melanoma cells could be accelerated in vitro and the in vivo tumour growth was surprisingly stimulated due to the decreased level of endogenous reactive oxygen species (ros). “The cellular respiration inhibition to HL-60 (myeloid) and Jurkat (lymphoid) cells was both concentration- and time-dependent, and the sba- 15-type msn inhibited cellular respiration at 25–500 mg ml however mcm-41-type msns had no noticeable effect on respiration rate due to the limited access to cellular mitochondria. In spite of the negligible cytotoxicity of msns.” MSNs with residual toxic surfactants such as CTAB which remained in the pore channels would exhibit remarkably magnified cytotoxicity. “MSNs used as nano-drug delivery system would be always desired to be non-cytotoxic, therefore it is necessary to completely remove toxic surfactants from the pore channels of msns prior to the drug loading.”

1.9.2 Blood compatibility

Vein injection is an important administration approach, by which drugs can efficiently be delivered to targeted cells and tissues. the blood compatibility of msns by investigating their hemolysis, coagulation, nonspecific protein binding and phagocytosis. The levels of prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (Fib) were measured to evaluate the coagulation behavior of msns. “The results indicated that neither APTT, PT nor Fib values of the sba-15 type msns exceeded their normal ranges in a broad concentration range of 50–500 mg mL_1, suggesting that the sba-15 type msns could not activate the intrinsic, extrinsic and common coagulation pathways. At low MSN concentrations of less than 100 mg mL_1, the hemolysis activity of the msns was confirmed to be almost invisible.”

1.9.3 Tissue compatibility

For the sustained release of drugs, msn_s are expected to stay at the targeted sites for a considerably long term. Therefore, the tissue compatibility of msn_s needs to be considered for biosafety. Recently, the tissue compatibility of msn was investigated by histopathological evaluation. “No pathological abnormality could be observed in both gross and microscopic histological examinations of various tissues including heart, liver, spleen, lung and kidney in one month after vein injection into mice, suggesting that the msn_s had not caused significant tissue toxicity and inflammation though they had not completely degraded.^17”

1.10 Antibacterial agents.

Antibacterial agent also known as antibiotics which the class of antimicrobial agent, used in treatment and prevention of infection of bacteria. Antibacterial drugs are derived from bacteria or molds or are synthesized de novo. Technically, “antibiotic” “refers only to antimicrobials derived from bacteria or molds but is often (including in the manual) used synonymously with “antibacterial drug.”Antibiotics sometimes interact with other drugs, raising or lowering serum levels of other drugs by increasing or decreasing their metabolism or by various other mechanisms.¹⁸In other world’s antibacterial agents are molecules that kill, or stop the growth of, microorganisms, including both bacteria and fungi.” According to mechanism of action, antibiotics divided in to two categories one is bacteriostatic and second bacteriorecidal. The definitions of “bacteriostatic” and “bactericidal” appear to be straight forward: “bacteriostatic” means that the agent prevents the growth of bacteria (i.e., it keeps them in the stationary phase of growth), and “bactericidal” means that it kills bacteria.¹⁹

The first antimicrobial agent in the world was salvarsan, a remedy for syphilis that was synthesized by Ehrlich in 1910. In 1935, sulfonamides were developed by Domagk and other researchers. “In 1928, Fleming discovered penicillin. Found that the growth of Staphylococcus aureus was inhibited in a zone surrounding a contaminated blue mold (a fungus from the Penicillium genus) in culture dishes, leading to the finding that a microorganism would produce substancesthat could inhibit the growth of other microorganisms .In 1944, streptomycin, an aminoglycoside antibiotic, was obtained from the soilbacterium Streptomyces griseus.” Thereafter, chloramphenicol, tetracycline, macrolide, and glycopeptides(e.g., vancomycin) were discovered from soil bacteria.The synthesized antimicrobial agent nalidixic acid, a quinolone antimicrobial drug, was obtained in 1962.²⁰

1.11 Mode of action of antibacterial agents

There is three type of mode of action present by which antibiotics are worked

a) Inhibition of Cell Wall Synthesis

b) Inhibition of Protein Synthesis (Translation)

c) Inhibition of nucleic acid synthesis

d) Antimetabolite activity

(a) Inhibition of cell wall synthesis

Bacterial cell contains a peptidoglycan cell wall in addition to the normal inner plasma membrane, surrounding the cellular contents. In other words, bacterial cells resemble the primitive plant cell structure. “In addition to these Gram-negative bacteria also has outer lipid bilayer. Some of the antibacterial compounds interfere with the cell wall synthesis by weakening the peptidoglycan structures in bacterial cell wall, by this integrity of bacterial cell wall structure weakens and eventually disrupts. Mammalian cells only have plasma membrane so these antibiotics specifically target only bacterial cells. That is these antibiotics do induce any negative effect on the host mammalian cells. The specificity of antibacterial compound β-lactam is by their ability to prevent the assembly of peptidoglycan layer via inhibiting transpeptidase enzyme activity.” Antibacterial compound β-lactam can be used against both Gram-positive and Gram-negative bacterial cells.

(b)Inhibition of protein synthesis

Some of the antibiotic compounds inhibit bacterial cell multiplication by inhibiting protein synthesis in them. Protein synthesis is a multi-step process. Majority of antibiotics inhibit the process that occurs in the 30S or 50S subunit of 70S bacterial ribosome, this in turn inhibits the protein biosynthesis.

(c) Inhibition of Nucleic acid Synthesis

This category of antibacterial compounds interferes in the synthesis of nucleic acid of bacterial cells. For example, compound quinonoles interfere with synthesis of dna molecule by inhibiting activity of enzyme topoisomerase. “This enzyme is involved in the dna (deoxy nucleic acid) replication. The second-generation quinolones like levofloxacin, norfloxacin and ciprofloxacin all can be used against both gram-positive and gram-negative bacteria. These compounds specifically inhibit the bacterial topoisomease II. Some antibiotics inhibit the action of enzyme rna polymerase, hence interfere with rna (ribonucleic acid) synthesis in the cells.²¹

(d)Anti metabolite activity

Any substance that competes with or inhibits the normal metabolic process, often by acting as an analogue of an essential metabolite known as anti-metabolite agents. Antimetabolites are also used as antibiotics. There are three main types of antimetabolite antibiotics. The first, antifolates impair the function of folic acid leading to disruption in the production of dna and rna. “The second type of antimetabolite antibiotics consist of pyrimidine analogues which mimic the structure of metabolic pyrimidines. Three nucleobases found in nucleic acids, cytosine (c), thymine (t), and uracil (u), are pyrimidine derivatives and the pyrimidine analogues disrupt their formation and consequently disrupt dna and rna synthesis.^22”

2. DRUG PROFILE^{23, 24}

2.1 Clarithromycin: Clarithromycin, a semisynthetic macrolide antibiotic derived from erythromycin, inhibits bacterial protein synthesis binding to the bacterial 50s ribosomal subunit. Binding inhibits peptidyl transferase activity and interferes with amino acid translocation during the translation and protein assembly process. Clarithromycin may be bacteriostatic or bactericidal depending on the organism and drug concentration.

2.2 Structure:

Figure2.1: Structure of clarithromycin

2.3 IUPAC name:

(3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-6-{[(2S,3R,4S,6R)-4-(dimethylamino)-        3-hydroxy-6-methyloxan-2-yl]oxy}-14-ethyl-12,13-dihydroxy-4-{[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy}-7-methoxy-3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-2,10-dion.

2.4 Molecular Formula: C₃₈H₆₉NO₁₃

 

2.5Molecular Weight: 748.0 da.

 

2.6 Category: Antibacterial agents, Protein synthesis inhibitors, Macrolides

2.7 Physical characteristics: white to off-white crystalline powder.

2.8 Solubility: Soluble in acetone, slightly soluble in dehydrated alcohol, in methanol and acetonitrile, practically insoluble in water.

2.9 Storage: Store in well closed container protected from light and should store below 30^.C.

2.10Melting point: 220^.C.

2.11 Mechanism of action: Clarithromycin is first metabolized to 14-OH clarithromycin, which is active and works synergistically with its parent compound. Like other macrolides, “it then penetrates bacteria cell wall and reversibly binds to domain V of the 23S ribosomal RNA of the 50S subunit of the bacteria ribosome, blocking translocation of aminoacyl transfer-RNA and polypeptide synthesis. Clarithromycin also inhibits the hepatic microsomal CYP3A4 isoenzyme and P-glycoprotein, an energy- dependantdrug efflux pump.”

2.12 Pharmacokinetics:

• Bioavailability : 50%
• Protein binding : 70% protein bound
• Metabolism : Hepatic
• Biological half-life : 3-4 hour
• Excretion: Urine (30%)

2.13 Pharmacodynamics: Clarithromycin is macrolide antibiotics whose spectrum of activity includes many gram-positive (Stephylococcus aureus, S. pneumonia, and S.  pyogenes)and gram-negative aerobic bacteria (Haemophilus influenza, H. parainfluenzae, and Moraxella catarrhalis), many anaerobic bacteria, some mycobateria and some other organism including Mycoplasma, Ureaplasma, Chlamydia, Toxoplasma and Borrelia . “Other aerobic bacteria that clarithromycin has activity against include C. pneumoniae and M.pneumoniae. Clarithromycin has an in-vitro activity that is similar or greater than that of erythromycin against erithrimycin susceptible organisms. Clarithromycin is usually bacteriostatic, but may be bactericidal depending on the organism and the drug concentration.”

2.14 Half-life: Elimination half-life is 3-4 hours

2.15 Dose: The recommended dose for adult in indications is 7.5 mg/kg bid.

2.18 Uses and administration: Claarithromycin is used to treat a wide variety of bacterial infections. This medication can also be used in combination with anti-ulcer medication to treat certain types of stomach ulcers. “It may also be used to prevent certain infection infections. Clarithromycin is known as a macrolide antibiotic. It works by stopping the growth of bacteria. This antibiotic treats only bacterial infections.” It will not works for viral infections (such as common cold, flu). Unnecessary use or misuse of any antibiotic can lead to its decreased effectiveness.

3. LITERATURE REVIEW

• Patil et al.(2011)⁵develop mesoporous silica nanoparticle with 2D hexagonal p6 symmetry and an average  particle size of 186 nm. The produced nanoparticle were used to load carbamazepine through a supercritical co2 process combined with various organic solvent.

• Yang et al. (2011)²⁵ prepared functionalized mesoporous silica material which is able to respond to envoirment changes, such as pH, redox potential, temperature and biomolecule.

• Chen et al.(2003)²⁶ reported “the preparation and characterization of porous hollow silica nanoparticles with diameter of 60-70nm with wall thickness approximately 10nm .Porous hollow mesoporous silica nanoparticle were synthesized by using CaCo₃ nanoparticles as the inorganic template.” Drug release profile “from porous hollow silica nanoparticles followed a three stage pattern and exhibid a delayed release effect.

 

• Nandiyatoet al. (2009)²⁷ reported spherical “mesoporous silica nanoparticles with tunable pore size and tunable “outer particle diameter in the nanometer range were successfully prepared in a water/oil phase using organic templates method.”This method involves the stimultaneoushydrolic condensation of tetraorthosilicate to form silica and polymerization of styrene in to polystyrene. An amino acid catalyst, octane hydrophobic-supporting reaction component, and cetyltrimethylammonium bromide surfactant were used in the preparation process. The final step in the method involved removal of the organic components by calcinations, yielding the mesoporous silica particles.

• Yuan et al. (2011)²⁸ developed Poly(acrylic acid) grafted mesoporous silica nanoparticles (PAA-MSNs) were prepared by a facile “graft on to strategy, i.e., the amidation” between PAA homopolymer and amino group functionalized MSNs. The resultant PAAMSNs were uniform spherical nanoparticles with a mean diameter of approximately 150 nm. The PAA-MSNs could be well dispersed in aqueous solution, which is favorable to be utilized as drug carriers to construct a pH-responsive controlled drug delivery system.

 

• Qaioet et al. (2009)^29”reportedcooperative self-assembly of “silica species and cationic surfactant cetyltrimethylammonium chloride (CTA⁺Cl^– orCTAC) and the formation of mesoporous silica nanoparticles occur following the hydrolysis and condensation of silica precursor TEOS in the solution.” The particle size can be controlled from 25 nm to 200 nm by adding suitable additive agents (e.g., inorganic bases, alcohols) which affect the hydrolysis and condensation of silica species. Results show that a certain acid-base buffer capacity of the reaction mixture in a range of pH 6-10 is essential for the formation of mesoporous silica nanoparticles in the TEOS-CTA⁺ system”.

• Nooneyet al.(2002)³⁰ Described one step procedure for the synthesis of spherical mesoporous silica,in “which the size of the particles is controlled over a range of diameters from 65 to 740 nm by varying the initial silicate/surfactant concentration under dilute conditions.. Synthesis using a charged template, cetyltrimethylammonium bromide, under aqueous conditions yielded particles of irregular spherical shape with highly ordered mesoporous channels.” Synthesis under ethanol/water cosolvent conditions yielded smooth spheres with a starburst mesopore structure extending from the center of the particle to the circumference.

• Sharmiladevi et al. (2016)³¹ Reported synthesis of mesoporous silica nanoparticles using the sol-gel method and to “determine the antibacterial activity of mesoporous silica nanoparticles and tetracycline loaded mesoporous silica nanoparticles. Procedure done by, the surfactant Cetyl Trimethyl Ammonium Bromide (CTAB) is initially dissolved in basic aqueous solution, and the mixture is vigorously stirred. Tetra Ethyl Ortho Silicate (TEOS) is added, and the solution is kept vigorous stirring for 6 h. After the reaction is complete, the as-synthesized” product is filtered and washed with deionized water. The antibacterial was tested using disc diffusion method and minimum bactericidal concentration (MBC).

 

• Lodha et al. (2012)³²discussed that Mesoporous silica nanoparticles (MSNs) are introduced as chemically and thermally stable nanomaterials with well-defined and controllable morphology and porosity. Silica nano-particles were synthesized by chemical methods from tetraethylorthosilicate (TEOS), methanol (CH3OH) and deionised water in the presence of sodium hydroxide as catalyst at 80°C temperature.

• Vivero-Escoto et al. (2010)²Reported the application of nanotechnology in the field of drug delivery has attracted much attention in the latest decades. Recent breakthroughs on the morphology control and surface functionalization of inorganic-based delivery vehicles, such as mesoporous silica nanoparticles (MSNs), “have brought new possibilities to this burgeoning area of research. The ability to functionalize the surface ofmesoporous-silica-based nanocarriers with stimuliresponsivegroups, nanoparticles, polymers, and proteinsthat work as caps and gatekeepers for controlled release ofvarious cargos.one of the main areas of interestin this fi eld is the development of site-specific drug delivery vehicles.”

 

• Jalvandi et al. (2015)³³ reported Nano fibrous materials use as carriers for clinical drugs but face the limitation of releasing the drugs in a burst fashion during use. “The aim of this study is to produce composite Nano fibrous mats with sustained release, using the broad spectrum antibiotic levofloxacin (LVF) as a model. Sustained release was achieved through two approaches.i.e.by firstly loading LVF into mesoporous” silica nanoparticles (MSN) and then incorporating the MSN in the core regions of poly (ecaprolactone)(PCL) nanofibres via core–shell electro spinning.

• Camporotondi et al. (2013)³⁴worked on nanoparticles containing antibiotics has demonstrated numerous advantages. “As the antibiotic is held into the nanoparticle, chemical composition and modifications on the NP’s surface enable to prolong, localize, target and have a protected drug interaction with the diseased tissue. In this way, higher antibiotic concentrations are attained in the targeted cells,” managing to reduce the frequency of the dosages taken, reducing the drug side effects and fluctuation in circulating levels, improving the overall pharmacokinetics.

 

4. RESEARCH ENVESIGED

Overuse of antibiotics has been described worldwide in both community and hospital settings particularly in developing countries.³³ Antibiotics resistance is one of the most important worldwide healthcare problems. Other factors contributing to the resistance include incorrect diagnosis, unnecessary prescriptions, improper use of antibiotics by patients and unfinished antibiotic prescription. “As a result, multiple drug resistance has appeared, mainly connected to hospital-associated infections and biofilm formation. The ability of certain strains of bacteria to withstand the effects of common antibiotics has led to find novel strategies for the treatment of infections associated to antimicrobial resistance and biofilm development in affected patients. The applications of inorganic nanoparticles to achieve good antimicrobial activity are growing fast. A large variety of nanomaterials for efficient antibiotic drug delivery have been developed and their efficacy has been demonstrated.” Due to high thermal and chemical stability, high surface area and good biocompatibility, the interest on silica nanoparticles as a system to deliver drugs such as antibiotics is increasing. Colloidal silica is a very versatile material as it can be prepared in a wide variety of forms and sizes, its surface is easy to modify and it can be obtained from relatively cheap precursors.³⁴

Mesoporous silica nanoparticles have been used to their low toxicity and high drug loading capacity, so they are used in controlled and target drug delivery system. “Due to strong Si-O bond, silica-based mesoporous nanoparticles are more stable to external response such as degradation and mechanical stress as compared to niosomes, liposomes, and dendrimers which inhibit the need of any external stabilization in the synthesis of MSNs.³ Mesoporous silica nanocarriers (MSNs) have recently brought new potential in nanomedicine due to their large surface area and pore volume, tunable, mesopore size and biocompatibility.”  The extensive mesoporosity and functionalized moieties on the pore surface make MSNs promising candidates to accomplish sustained and/or controlled drug delivery.

Therefore mesoporous silica nanoparticles were selected as drug delivery system to deliver antibacterial agent through oral route. “Mesoporous silica nanoparticles contain hollow space which could contain the larger amount of drug. Due to the large surface area of mesoporous silica nanoparticle release rate of the drug is also increases. Mesoporous silica nanoparticle showed sustained release drug delivery due to their sustained release property. The main aim of our project is development antibacterial loaded mesoporous silica nanoparticle for sustained release of drug and avoiding resistance developed due to antibiotic. In addition of being biocompatible, msn will be highly suitable to drug delivery because of its permeation enhancement, mucoahesiveness and its ability to perform sustained and controlled release.” Mesoporous silica nanoparticle is very stable in acid, polyelectrolyte multilayer coated silica would significantly retard leach in acidic envoirment. “Therefore in the gastric environment control on the drug release rate will be more effective on standolane basis for msn.³⁵As the antibiotic will be entrapped into the nanoparticle, chemical composition and modifications on the NP’s surface enable to prolong, localize,” target and have a protected drug interaction with the diseased tissue. In this way, higher antibiotic concentrations will attained in the targeted cells, managing to reduce the frequency of the dosages taken, reducing the drug side effects and fluctuation in circulating levels, thus improving the overall p

 

5. PLAN OF WORK

 

1. Literature review

 

2. Selection of drug

• Criteria of selection of drug for oral drug delivery

 

3. Preformulation studies

• Test of identification

• Physical Appearance
• Melting point determination
• FTIR spectra of drug
• Solubility studies
• Partition coefficient determination

• Quantitative estimation of drug

4.      Formulation of mesoporous silica nanoparticle.

 

 

5. Characterization of prepared mesoporous nanoparticle.

• Surface morphology (SEM studies)
• Particle size analysis(zeta sizer)
• X-ray diffraction
• FTIR spectra of mesoporous silica nanoparticle
• Drug loading eficiency
• In vitro Release studies
• Ex vivo antibacterial study of mesoporous silica nanoparticle.

 

6. Result and discussion

 

7. Conclusion

 

6. PREFORMULATION STUDY

6.1 TEST OF IDENTIFICATION

1. Physical appearance: A fine, white crystalline powder.
2. Melting point: melting point of clarithromycin was found to be 217-220⁰c by using melting point apparatus.
3. I.R. spectra: Identification of drug was done by its IR Spectra. The Infra red spectral assignment of clarithromycin was obtained by FTIR( Jasco 470 plus) “The sample IR spectrum of doxorubicin hydrochloride was interpreted and matched with the reference IR spectrum given in Table 6.1,” and it was inferred that the sample compound contains all the peaks as obtained in the reference sample of the drug.

Figure 6.1 Reference I.R. spectrum of clarithromycin

4. Solubility profile: Solubility of clarithromycin was tested in various aqueous and organic solvents. A specific amount of drug was dissolved in specific amount of different solvents at room temperature and was observed only by visual inspection. Result are given in Table:

Table 6.1 Solubility profile of clarithromycin.

f) Partition coefficient: Partition coefficient is a measure of drug lipophilicity and an indication of its ability to cross the biological membrane reference Partition coefficient is defined as the ratio of unionized drug distributed between the organic and aqueous phase at equilibrium.

P_o/w  =  (C _oil/Water) _Equilibrium

clarithromycin was weighed (10mg) and dissolved in 10ml each of n-octanol and PBS (pH7.4). The mixture was shaken properly for 24 hrs in a separating funnel. The aqueous phase was separated out and assayed for amount of drug after proper dilution.

3.2 Quantitative estimation of drug:

The spectrophotometric method Preparation of calibration curves: The standard curves of clarithromycin was prepared in phosphate buffer (pH 7.4) and 1.2 HCl acid buffer using UV-Visible spectrophotometric analysis.

• Prepration of phosphate buffer (7.4))-  To prepare phosphate buffer, 6.8 gm of sodium acetate and 3 ml of glacial acetic acid were dissolved in sufficient water to produce 500 ml and pH was adjusted accordingly.

 

• Determination of absorption maxima ( ƛ max) in 7.4 phosphate buffer

100 mg of clarithromycin was weighed accurately and transferred to a 100 ml volumetric     flask, and the volume was adjusted to the mark with the buffer (pH 7.4). From above  solutions of 0.1 ml was  transferred to 10 ml volumetric flasks, and make up the volume up to mark. Resulting solution take 2ml and 1ml of BCG (2% w/v) and extract with chloroform, Pipette out aqueous layer and scanned over UV range (400-800nm), maximum absorbance was found at    Lambda max 666.0 nm

• Preparation of calibration curve of clarithromycin in 7.4 phosphate buffer

• 10mg of clarithromycin was weighed accurately and “transferred to separate 10ml volumetric flask, “and the volume was adjusted to the mark with the buffer (pH 7.4) to give a stock solution of 1000 µg/ml.”
•  From stock solutions of clarithromycin 1 ml was taken and diluted up to 10 ml for make 100 µg/ml substock solution.
•  from this solution 1.0, 2.0, 3.0, 4.0, 5.0 ml solutions were transferred to 10ml volumetric flasks and make up the volume up to 10 ml with buffer (pH 7.4), gives  standard drug solution of 10, 20, 30, 40, 50 µg/ ml concentration.”
• Resulting solution take 2ml and 1ml of BCG (2% w/v) and extract with 3ml chloroform, Pipette out aqueous layer and absorbance was taken at 666.0 nm.³⁸

Table 6.2 Standard curve data of clarithromycinin (7.4 pH)

 

Fgure 6.2 Calibration curve of clarithromycin in 7.4 phosphate buffer saline.

• Preparation of 1.2 HCl acidic buffer :to prepare1.2 HCl acid buffer,add 125 ml of 0.2 M KCl with 212.5 ml of 0.2 M hcl and volume make up to 500 ml with distilled water.

 

• Determination of absorption maxima ( ƛ max) in 1.2 pH HCl buffer

100 mg of clarithromycin was weighed accurately and transferred to a 100 ml volumetric flask,   and the volume was adjusted to the mark with the buffer (pH 1.2). From above  solutions of 0.1 ml was  transferred to 10 ml volumetric flasks, and make up the volume up to mark. Resulting solution take 2ml and 1ml of BCG (2% w/v) and extract with chloroform, Pipette out aqueous layer and scanned over UV range (400-800nm), maximum absorbance was found at Lambda max 616.0 nm.

• Preparation of calibration curve of clarithromycin in 1.2 pH HCl buffer

• 10mg of Clarithromycin was weighed accurately and transferred to separate 10ml volumetric flask, and the volume was adjusted to the mark with the buffer (pH 1.2) to give a stock solution of 1000 µg/ml.
• From stock solutions of clarithromycin 1 ml was taken and diluted up to 10 ml for make 100 µg/ml sub stock solution.
• “from this solution 1.0, 2.0, 3.0, 4.0, 5.0 ml solutions were transferred to 10ml volumetric flasks and make up the volume up to 10 ml with buffer (pH 1.2), gives  standard drug solution of 10, 20, 30, 40, 50 µg/ ml concentration.”
• Resulting solution take 2ml and 1ml of BCG (2% w/v) and extract with 3ml chloroform, Pipette out aqueous layer and absorbance was taken at 616.0 nm.³⁸

Table 6.3 Standard curve data of clarithromycinin ( 1.2pH)

 

          . .

Figure 6.3 calibration curve of clarithromycin in 1.2 pH HCl buffer

7. EXPERIMENTAL WORK

7.1 PREPARATION OF MESOPOROUS SILICA NANOPARTICLES    

• CTAB (0.5g) 2.0M NaOH (1.75ml) and deionised water (120g) were heated at 80°C for 30 minutes to reach a PH of 12.3.
• this clear solution TEOS (2.34gm) 2.5ml is rapidly injected via injection under vigorous stirring.
• “a white precipitation will be observed within three minutes of stirring at 550 rpm.
• The reaction temperature is maintained at 80°C for 2 hours.
• The product is isolated by hot filtration washed with copious amount of water and methanol (5ml each for three times).

7.2 ACID EXTRACTION OF MESOPOROUS SILICA NANOPARTICLE

• An acid extraction was performed in a methanol (100 mL) mixture of concentrated hydrochloric acid (1.0 mL) and as-made materials (1.0 g) at 60C for 6 h.

• The surfactant-removed solid products were filtered and washed with water and methanol, and then dried under vacuum.”

7.3 LOADING METHOD OF CLARITHROMYCIN IN MESOPOROUS       SILICA NANOPARTICLE       

• For this process take nanoparticle and drug in 1:2 ratio. Clarithromycin disperse  with the 7.4 pH buffer saline by 10 min sonication.
• After sonication add nanoparticle and stirrer the drug and nanoparticle system upto 24 hrs.
• Then centrifuge the drug system mixture at 20000 rpm. 

 

 

 

7.4 CHARECTERIZATION OF THE MESOPOROUS SILICA NANOPARTICLE

Charecterizaion of mesoporous silica nanoparticle is charecterize by following method-

7.4.1 Particle size ananlysis

The mesoporous silica nanoparticle was characterized for particle size using Zeta Sizer, Malvern Instrument Ltd. UK ZS-90. Take some drop of final product before the acid extraction, dissolved with deionised water and cuvet filled with the deionised water was used for particle analysis.

7.4.2 Scanning electron microscopy

SEM study was done by FEI NOVA NANO SEM (450, NETHERLAND). For sample preparation, sample are mounted on the stub of gold with double adhesive tape and then observed in the microscope.

7.4.3 LA-XRD of mesoporous silica nanoparticle

Mesoporous silica nanoparticle show peak in the low angle which is done by Powder     diffraction data can be collected using either transmission or reflection geometry. Because the particles in the powder sample are randomly oriented, “these two methods will yield the same data. In the MRL, x-ray facility, powder diffraction data are measured using the Phillips XPERT MPD diffractometer, which measures data in reflection mode and is used mostly with solid   samples. Powder XRD study was performed by XRD, RIGAKU – JAPAN (MINIFLEX) with a scanning range of 0 to 10⁰ (2θ) for the structural characterization of mesoporous silica nanoparticle.

7.4.4 Fourier transform-infrared spectroscopy

The IR spectra mesoporous silica nanoparticle and clarithromycin loaded msn were obtained using Prestige-21,” FT-IR spectrophotometer, Shimadzu Corp. Japan. One mg of each sample was finely grounded with KBr (Merck) into a fine powder separately in motor pestle and poured into the sample holder by means of a spatula and scanned over a wave number of 4000-650 cm^-1 and characteristic bands were recorded.

     7.4.5 DRUG LOADING EFFICIENCY

To identify the drug loading efficiency of the system, centrifuge the system drug mixture at           20000 rpm and collect supernatant. Supernatant was observed spectrophotometicaly at 666.0 nm meter and the clarithromycin loading efficiency was calculated using the following formula:

7.4.6 In vitro release study

In vitro release study of clarithromycin loaded msoporoussilica nanoparticle performed in phosphate buffer saline (7.4pH) and 1.2pH HCl acid buffer as the recipient media in order to mimic physiological and gastric pH at 37± 0.5℃ “using dialysis membrane packet in dissolution apparatus. Aliquots (1 ml) were withdrawn at pre-determined time intervals (1, 2, 3, 4, 5, 6,7,8 and 24 hrs.) and after withdrawal of aliquots the recipient medium was replenished with the same volume of buffer solution (1 ml). The concentration of drug was determined by UV-Visible spectrophotometry.” The in vitro release studies data so obtained was fitted to various release kinetic models such as zero order, first order, and Higuchi and Korsemeyer Peppas models in order to determine the release mechanism from the mesoporous silica nanoparticle. Data is presented in the table 8.8.

7.4.6.1 Data interpretation

The in vitro release of clarithromycin loaded mesoporous silica nanoparticle was performed in phosphate buffer (pH 7.4) and HCl buffer (pH 1.2) as recipient media using dialysis membrane packet at 37± 0.5°C.

The results of in vitro release studies were subjected to the following kinetic models:

• Zero order (cumulative per cent of drug release v/s time)
• First order (log cumulative per cent of drug remaining v/s time)
• Higuchi square root law (cumulative per cent of drug release v/s square root of time)
• Korsemeyer Peppas model (log of cumulative per cent of drug release v/s log time)

The release data was plotted according to the following equation:

• Zero order: M = M0 – K0t
• First order: log C = Log C0 – Kt/2.303
• Higuchi square root law: Q = Ktn
• Korsemeyer’s Peppas model: Mt/M∞ = Ktn

Where M, C and Q is the amount of drug release at time t, M0 & C0 is the total amount of drug. K0, K & k are corresponding rate constants.  In case of Korsemeyer’s Peppas model, “Mt/M∞” is the fractional drug release at time, “K” is the rate constant and “n” is the release exponent. The value of “n” is calculated from Korsemeyer’s Peppas equation. It is used to interpret different mechanisms of drug release.

7.4.7 Antimicrobial activity of clarithromycin loaded mesoporous silica nano particle

The anti-microbial activity of drug loaded mesoporous silica nanoparticle and standard antibiotic (clarithromycin) was determined by disk-diffusion method and agar dilution method microorganism are select for the anti-microbial activity was

1.  S.aureus,
2. S.mutans,
3.  E.coli
4.  P.vulgais

When a filter paper disc impregnated with a chemical is placed on agar the chemical will diffusion from the disc into the agar. “This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as a “zone of inhibition”. “After incubation, zone diameter is measured to the nearest whole millimeter at the point where in there is a prominent reduction of 80% growth. Software analyzed bacterial culture were grow over night in liquid broth then bacterial culture was spread on NAM agar medium plats.” Labeled the back of the NAM plats through marker. 50, 100, 150, 200, 250, 300(µg) Placed the 6mm what’s man filter paper No. disc of different concentration of different drug sample. (50, 100, 150, 200, 250, 300(µg)).”Incubate at 37°C for 12-14 hrs. After incubation the zone of inhibition in mm was measured by zonal scale.

8. Result and discussion

8.1 Preformulation studies:

A) Identification of drug:

      Physical appearance: clarithromycin was found fine, white, crystalline powder.³⁹

      Melting point: ⁴⁰

Table 7.1 Melting point

 

Solubility studies: The solubility of clarithromycin was checked by visual inspection in different solvents. The result was obtained in the following Table:

Table. 8.2 solubility study

Partition coefficient: Partition coefficient of clarithromycin was determined in a 1:1 mixture of n-octanol and 7.4pH phosphate buffer. The resulting partition coefficient was calculated and reported:

Table 8.3.Partition coefficient

8.2. PREPARATION OF MESOPOROUS SILICA NANOPARTICLES^45,1,31  

MCM 41 typemesoporous silica nanoparticle have been produced by the sol-gel method which include surfactant like CTAB (cetyltrimethyl ammonium bromide) as liquid crystal templating,”TEOS (tetra ethyl orthosilicate) as the silica precursor,and NaOH use as catalyst. This process require two step hydrolysis and condantation.synthesis of mesoporous silica nanoparticle using sol-gel method was confirmed y the formation of white colour formation of adding TEOS”. Synthesis of msn is performed at low concentration of CTAB to make assembly of mesophase by interaction between the cationic surfactant and developing anionic oligomer orthosilic acid.

8.3. CHARECTERIZATION OF THE MESOPOROUS SILICA NANOPARTICLE

a) Particle size analysis

The particle size of mesoporous silica nanoparticle was determined by Zetasizer (Malvern).size of mesoporous silica nanoparticle is198 nm. Report is attached in the annexures.

b) SCANNING ELECTRON MICROSCOPY

SEM study was done by FEI NOVA NANO SEM (450, NETHERLAND) which image is following:

 

Figure 8.1 Microscopic image of msn at 1µm

Figure 8.2 Microscopic image of msn at 500nm

c) LA-XRD OF MESOPOROUS SILICA NANOPARTICLE

Low angle X-ra diffraction done in by XRD, RIGAKU – JAPAN (MINIFLEX) at 2ϴ scanning range 0 to 5 were show the sharp peak on the 2.059⁰ angle. peak on 2.059⁰ angle define the MCM_41 type of mesoporous silica nanoparticle

Figure8.3 LA Xrd of mesoporous silica nanoparticle

d) Fourier Transform-Infrared Spectroscopy:

FT-IR analysis of clarithromycin, mesoporous silica nanoparticle and clarithromycin loaded mesoporous silica nanoparticle was performed and interpreted. The data are shown in the following Figure.

Figure 8.4.  FT-IR of clarithromycin

Table 8.4 Characteristic IR absorption bands of clarithromycin

Figure 8.5.FT-IR of mesoporous silica nanoparticle

Table7.5. Characteristic IR absorption bands of mesoporous silica nanoparticle

 

The peak of 1224.80 cm^-1 show presence of Si-o-Si and peak 3213 cm^-1show presence of Si-OH group which are responsible for silia nanoparticle formation.

Figure 8.6.FT-IR of drug loaded mesoporous silica nanoparticle

Table 8.6 Characteristic IR absorption bands of mesoporous silica nanoparticle

Peak 1236.46 cm^-1, 3483.44 cm^-1 the presence “of Si-O-Si group, SiOH which show the silica nanoparticle and peak 1456.57 cm^-1show the presence of N-CH3 group which is important functional group of clarithromycin. Ketone, O-C-O- streaming and Alkane stretching found at1691.57, 1172.72 and 2970.38 cm^-1.”following interpretation show the interaction between silica nanoparticle and clarithromycin drug.

e) Loading efficiency:

The loading efficiency of clarithromycin was calculated in PBS (pH 7.4) at 666.0 nm using UV/Visible spectrophotometer and was found to be 96 % respectively.

8.5 In vitro release studies:

Release study of clarithromycin loaded mesoporous silica nanoparticle was performed in PBS pH 7.4 and HCl buffer pH 1.2 over a period of 24 hrs. and the data was presented in the following Table:

Table 8.8 in vitro release data

Data treatment:

To determine the mechanism of the drug release from the drug loaded formulations, the in-vitro release data were treated using the following mathematical models. The release data were plotted according to the following equations:

• Zero order : M = M₀-K₀t
• First order : Log C = Log C₀-Kt/2.303
• Higuchi square root law: Q = Ktⁿ 
• Korsemeyer Peppas model: M_t/M_∞ = ktⁿ 

where M, C and Q is the amount of drug released at time t, M₀, and C₀ is the total amount drug and K0, K and k are corresponding rate constants. In case of Korsemeyer Peppas model, Mt/M∞ is the fractional drug released at time t, k is the constant incorporating the properties of macromolecular polymeric systems and the drug, and n is a kinetic constant which is used to characterize the transport mechanism. The exponent ‘n’ determines the release mechanism as given in Table 8.9. From the above equations the correlation coefficient and exponential (n) values for different formulations have been calculated to identify the drug release mechanism

Table 8.9: Release exponent (n) values

Table.8.10: Cumulative % drug release and log cumulative % drug release profiles of clarithromycin loaded mesoporous silica nanoparticle

 

Figure. 8.7. Zero order release graph of Clarithromycin/Mesoporous silica nanoparticle in PBS 7.4 pH and HCl buffer 1.2 pH

Figure. 8.8. First order release graph Clarithromycin/Mesoporous silica nanoparticle in PBS7.4 pH and HCl buffer 1.2 pH

Figure. 8.9 Higuchi diffusion release graph Clarithromycin/Mesoporous silica nanoparticle in PBS7.4 pH and HCl buffer 1.2 pH

 

Figure. 8.10. Korsemeyer’s Peppas release graph of Clarithromycin/Mesoporous silica nanoparticle in PBS7.4 pH and HCl buffer 1.2 pH

“The release of drug (clarithromycin) from mesoporous silica nanoparticle was checked in both PBS 7.4 and HCl buffer1.2pH that mimic the normal physiological milieu and stomach acid envoirment. In pH 7.4, 98.46% and 89.24 % of the entrapped drug was found to be released from mesoporoussilica nanoparticle after 24 hr of exposure to release medium.”

The correlation coefficient values of different release kinetic models were compared for PBS 7.4 and HCl buffer 1.2. The data is presented in the following Table:

Table 8.11: Comparison of correlation values of different release kinetic models

From the above data, it can be concluded that drug loaded mesoporous silica nanoparticle follow the Higuchi equation because its r² value are close to unity. This explains that the drug release mechanism follows Higuchi model, in which the drug release occurs as a result of diffusion from the mesoporous silica nanoparticle, hence it was selected as the best fit model.

8.6. Antimicrobial activity of clarithromycin loaded mesoporous silica nano particle

Antimicrobial activity of mesoporous silica nanoparticle was performed on the following bacteria S.aureus, S.mutans, E.coli and P.vulgais wich result are following:

8.6.1 Zone of inhibition

Table 8.12 zone of inhibition

Figure 8.11 zone of inhibition

8.6.2 Minimum effective concentration study

Table 8.13 minimum effective concentrations

Figure 8.12 minimum effective concentrations

10. Conclusion

In summary mesoporous silica nanoparticle with the controlled particle size (198nm) were done by using the softgel method. In which TEOS (Tetraorthosilicate) use as silica precursor and CTAB (cetyl tri ammonium bromide) use as the surfactant. Clarithromycin was load in in maner of 1: 2 where system take one part nad drug take 2 part. “mesoporous silica nanopwrticle have the high loading capacity by which 96% drug will be load on the nanoparticle.LA-Xrd of the prepared mesoporous silica nanoparticle shows the peak on 2.059^o between the range of 1^o to 5^o of 2ϴ. Peak on 2.059^o show the MCM-41type silica nanoparticle. IN FTIR spectra of the mesoporous silica nanoparticle show the peak in 1224.80 cm^-1,” show the presence of Si-O-Si group which is responsible for formation of the mesoporous silica nanoparticle.

“Release study of the clarithromycin loaded mesporous silica nanoparticle was performed in Phosphate buffer 7.4pH and acidic buffer 1.2pH. release of clarithromycin  from mesoporous silica nanoparticle were occur in  sustained manner, approx. 98.46 % drug was release in 24 hour in pH 7.4 and 89.24 % drug was release in HCl buffer 1.2 the 24 hour.

Antimicrobial activity of clarithromycin loaded mesoporous silica nanoparticle was performed against bacteria s.aureus, s.mutans, e.coli and p.vulgais.” Zone of inhibition is occurring very well against standard formulation. In bacteria E.coli zone of inhibition show the good results.