DEVELOPMENT AND CHARACTERISATION OF FLOATING MICROSPHERES OF FAMOTIDINE FOR THE TREATMENT OF PEPTIC ULCER

Drug delivery system

Drug delivery systems (DDSs) are pharmaceutical formulations or devices that help in achieving targeted delivery and/or controlled release (CR) of therapeutic agents in our body. Following administration, the DDSs liberate the active ingredients, and subsequently, the bioactive molecules are transported across various biological barriers to reach the site of action. The scientists have contributed substantially to understand the role of different physiological barriers in efficient transport of drugs in the circulatory system and drug movement through cells and tissues. In addition, their significant contribution to the development of a number of new modes of drug delivery has entered clinical practice. Despite a significant advancement in the process of new drug design and discovery, many drugs have unacceptable side effects due to interaction of the drug with parts of the body that are not the target for the drug. Sometimes, side effects occur depending on the medication, the mode of delivery, and response from our body. The buildup of high blood plasma drug concentration due to accumulation of drug from repeated administration of conventional DDS may lead to untoward side effects. Hence, the attempts must be made to afford better patient compliance effect from the reduction in the number and frequency of doses needed to maintain the desired therapeutic responses. These side effects can vary greatly from person to person in type and severity. The method by which the drug is delivered can have a significant impact on its efficacy (Garg et al., 2020)

1.2 Novel drug delivery system (NDDS)

In the past few decades, considerable attention has been concentrated on the evolution of a novel drug delivery system (NDDS) for herbal drugs. Conventional dosage forms, including prolonged-release dosage forms, are unable to satisfy for both holding the drug component at a distinct rate as per directed by the requirements of the body, all through the period of treatment, as well as directing the phytoconstituents to their desired target site to obtain an utmost therapeutic response. In phytoformulation research, developing nano-sized dosage forms (polymeric nanoparticles and nanocapsules, liposomes, solid lipid nanoparticles, phytosomes, and nanoemulsion) has a number of advantages for herbal drugs, including enhancement of solubility and bioavailability, protection from toxicity, enhancement of pharmacological activity, enhancement of stability, improving tissue macrophage distribution, sustained delivery, and protection from physical and chemical degradation. Thus, the nano-sized NDDSs of herbal drugs have a potential future for enhancing the activity and overcoming problems associated with the plant medicines. Liposomes, which are biodegradable and essentially nontoxic vehicles, can encapsulate both hydrophilic and hydrophobic materials (Medina et al., 2004).With the progress in all domains of science and engineering, the dosage forms have developed from simple mixtures and pills to highly sophisticated technology, intensive drug delivery systems, which are known as NDDSs (Bhagwat R et al., 2013).

1.3 Microspheres

Microspheres are polymeric micron range particles with sizes from 1 to 1000 µm. These microspheres are used for drug delivery, wherein the drug can be encapsulated or entrapped form. Based on the polymeric composition of microspheres, they can be classified into two types: natural and synthetic. Natural polymers include carbohydrates (e.g., chitosan, agarose, starch, alginate) and proteins (e.g., albumin, gelatin), while synthetic polymers include nonbiodegradable (e.g., polymethyl methacrylate, epoxy polymers) and biodegradable (polylactic acid/polyglycolic lactic acid) (Prasad, Gupta, Devanna, & Jayasurya, 2014). Microspheres in drug delivery are used for targeted as well as prolonged drug release in the diseased area. It also protects the unstable or pH-sensitive drugs before and after the administration. Microspheres are classified into four different types:

1.4 Types of Microsphere

1. Bioadhesive microspheres

2. Magnetic microspheres

3. Floating microspheres

4. Radioactive microspheres

5. Polymeric microspheres

i) Biodegradable polymeric microspheres

ii) Synthetic polymeric microspheres

1.4.1 Bioadhesive microspheres

These types of microsphere exhibit mucoadhesive property, which allows the drug coated on the surface of the polymer to stick to the targeted organ, resulting in prolonged delivery of the therapeutic agents to the diseased site.

1.4.2 Magnetic microspheres

These microspheres consist of magnetic particles and have a potential to be used for targeted delivery of drugs. Such microspheres can be used for both diagnostic purposes and drug delivery. The drugs encapsulated or coated within these particles can be targeted to the diseased area using an external magnetic source (Joshi et al., 2011). They are utilized very commonly for magnetic hyperthermia in tumor tissues.

1.4.3 Floating microspheres

Floating microspheres are meant to release the drugs loaded in them in gastric content. The bulk density of these drug-loaded microspheres should be kept lower than the gastric juice so that they can float on the surface, thereby having a prolonged drug release.

1.4.4 Radioactive microspheres

Radioactive particles (10–30 µm) are used

for the therapeutic purpose by directly injecting in the veins that are linked to the targeted organ or tissue. These radioactive particles emit three different types of waves: ? emitters, ? emitters, and ? emitters. There are different techniques to prepare such microspheres, and the process of preparation depends on the size of microspheres, route of administration, duration of drug cross-linking, duration of drug release, etc.

1.6 Application of Microspheres in Pharmaceutical Industry

1.6.1 Microspheres in Vaccine Delivery

The precondition of a vaccine is safety toward the microbes and its harmful components. An ideal vaccine should satisfy this same necessity of effectiveness, protection, affordability in application and charge. The aspect of protection and avoidance of severe effects is complicated. The aspect of safeness and the extent of the manufacturing of antibody responses are intently linked to mode of application.  Biodegradable delivery technology for vaccines which are provided by intravenous path  may resolve  the  shortcoming of  this same  conventional vaccines.  The involvement  in parenteral  (subcutaneous,  intramuscular,  intradermal) carrier  exists  even  though  those  who  offer  significant benefits.

1.6.2 Ophthalmic Drug Delivery

Microspheres developed using polymer exhibits favorable biological behavior such as bioadhesion, permeability-enhancing properties, and interesting physico-chemical characteristics, which make it a unique material for the design of ocular drug delivery vehicles. Eg. Chitosan, Alginate, Gelatin. 

1.6.3 Oral drug delivery

The potential of polymer matrix  usually contains diazepam like  an oral  drug delivery  has been  evaluated through  rabbits.  Its  findings showed  that  even  a  film consisting  of  a  1:0.5  drug-polymer  combination  may have been an effectual dosage form which is comparable to  commercial  tablet  formulations.  The capacity  of polymer  to  establish films  could  allow  use  in  the formulation of film dosage forms, as an option with drug tablets.  The pH  sensitivity,  combined  with  both  the reactions  of  the  main  amine  groups,  start  making polymer  a  distinctive  polymer  for  oral  drug  delivery applications.

1.6.4 Gene delivery

Genotype drug delivery involves viral vectors, nonionic liposomes, polycation complexes,  and  microcapsules technologies.  Viral vectors are beneficial for genotype delivery even though those who are extremely efficient and also have a broad variety of cell goals.  Even so,  if used  in  vivo  they  trigger  immune  responses  and pathogenic  effects.  To  resolve  the  restrictions of  viral vectors,  nonviral  delivery systems  have  been regarded for  gene  therapy.  Nonviral  delivery system  does  have benefits these as simplicity of  preparation, cell  / tissue targeting, reduced immune system, unrestricted plasmid size,  as  well  as  largescale replicable production. Polymer Will be used as a transporter of DNA for gene delivery applications.

1.6.5 Nasal drug delivery

Polymer based drug delivery systems, such as microspheres, liposomes and gels have been demonstrated to have good bioadhesive characteristics and swell easily when in contact with  the nasal mucosa increasing the bioavailability and residence time of the drugs to the nasal route. Eg.Starch, Dextran, Albumin, Chitosan+Gelatin (Virmani and Gupta, 2017).

1.7 Advantages of microspheres

• Particle size reduction for enhancing solubility of the poorly soluble drug (Mohan M et al., 2014).
• Provide constant and prolonged therapeutic effect. provide constant drug concentration in blood thereby increasing patent compliance,
• Decrease dose and toxicity
• Protect the drug from enzymatic and photolytic cleavage hence found to be best for drug delivery of protein.
• Reduce the dosing frequency and thereby improve the patient compliance
• Better drug utilization will improve the bioavailability and reduce the incidence or intensity of adverse effects.
• Microsphere morphology allows a controllable variability in degradation and drug release.
• Convert liquid to solid form & to mask the bitter taste.
• Protects the GIT from irritant effects of the drug.
• Biodegradable microspheres have the advantage over large polymer implants in that they do not require surgical procedures for implantation and removal.
• Controlled release delivery biodegradable microspheres are used to control drug release rates thereby decreasing toxic side effects, and eliminating the inconvenience of repeated injections.

1.8 Disadvantages

Drug discharge from measurements structure differs with an assortment of variables like inalienable and outward  factors, food, and the pace of move through the gut The distinction in the delivery rate starting with one portion then onto the next Controlled  discharge  arrangement  normally  having a  high  amount  of medications contains  veracity and any    vanishing    of    the    measurements    structure discharge trademark  it  might  show  to  bring  about the  development  of  conceivable poisonousness  and treatment disappointment Such measurement structures ought not be compacted or bitten Short drug stacking (limit of half) for the controlled delivery measurement structure, Once    managed    it    is    difficult    to    eliminate    the transporter totally from the body, Parental conveyance of  microsphere may  act  together or structure edifices with the blood segment (Kunchu et al;2010)

1.9 Limitation

Some of the disadvantages were found to be as follows (Kunchu et al; 2010). 

1. The costs of the materials and processing of the controlled release preparation are substantially higher than those of standard formulations. 

2. The fate of polymer matrix and its effect on the environment. 

3. The fate of polymer additives such as plasticizers, stabilizers, antioxidants and fillers. 

4. Reproducibility is less. 

5. Process conditions like change in temperature, pH, solvent addition, and evaporation/agitation may influence the stability of core particles to be encapsulated. 

6. The environmental impact of the degradation products of the polymer matrix produced in response to heat, hydrolysis, oxidation, solar radiation or biological agents. (Mohan M et al.,2014)

1.11 Methods used in microsphere preparation

Choosing the method depends primarily on the Character of a polymer  been using,  the drug, the factors  equivocally determined  by  many  formulations  and  technological factors as  the size of the  particles requirement,  and the drug or protein should not be significantly impacted by the process, the reproducibility of the release profile and the method, there should be no stability Issue, in relation to the finished product. The various types of procedures used to prepare the microspheres using hydrophobic and hydrophilic polymers as matrix materials.

The capacity to integrate medication doses which are relatively small. Stability of preparation after synthesis with a shelf spam which is clinically acceptable.

Controlled particle size and dispensability for injection in the aqueous vehicles. Effective reagent release with strong control over a large time-scale. Biocompatibility of controllable  biodegradability and chemical alteration response.

1.11.1 Wax coating and hot melt

Wax used to encapsulate the main components, by dissolving or dispersing the product in melted wax. The waxy paste or mixture, such as frozen liquid paraffin, is released by high intensity blending with cold water. The water is heated up for at least an hour. The substance is stirred up for at least 1 hour.  Then the external layer (liquid paraffin) is decanted and the microspheres  are immersed  in  a  non-miscible  solvent  and dry air  is required to dry.  For the surface ingredients, carnauba wax  and  beeswax  can  be  used, and  both  should be combined to obtain desirable characteristics. 

1.11.2 Spray drying technique

This was used to prepare polymer microsphere mixed charged with drug.  This  requires  dispersing  the  raw substance into liquefied coating liquid, and then spraying the  mixture  into  the  air  for  surface  solidification accompanied  by  rapid  solvent  evaporation. Organic solvent and polymer solution are formulated and sprayed in various weight ratios and drug in specific laboratory conditions producing microspheres filled with medications. This is fast but may lose crystalinity due to rapid drying (Kunchu et al., 2010).

1.11.3 Coacervation

This method  is  a  straight  forward  separation  of macromolecular  fluid  into  two immiscible  types  of material,  a  thick  coacervate  layer,  comparatively condensed  in macromolecules, and  a  distilled layer  of equilibria.  This method is referred to as basic coacervation, in the presence of just one macromolecule. If two or more opposite-charge macromolecules are involved, they are considered complex coacervation. The former is caused by  specific factors  including temperature  shift, Using  non-solvent  or  micro-ions contributing  to  dehydration  in  macromolecules,  since they facilitate interactions between polymer and polymer through  polymer  solvent  interactions.  This can be engineered to generate different properties on microsphere (Kunchu et al; 2010).

1.11.4 Solvent evaporation)

The  method  of  solvent  evaporation  has  also  been extensively  used  to  preparation  of PLA  and  PLGA microspheres which contain many various drugs. Several variables  were  identified  that  can  significantly  affect microspheric characteristics, such  as solubility  of drug, internal  morphology,  type  of  solvent,  diffusion  rate  , temperature,  polymer composition  as well  as viscosity, and  drug  loading.  The  efficacy  of  the  solvent evaporation system  to create  microspheres relies on the effective  entanglement of  the active substance  into the particles,  and  therefore  this  procedure  is  particularly efficient with drugs that are either insoluble or partially soluble in the liquid medium that constitutes the constant phase (Kunchu et al., 2010). 

1.11.5 Precipitation

It is a modification of the form of evaporation.  The emulsion is polar droplets scattered over a non-polar medium.  The use of a co-solvent can extract solvent from the droplets.  The  subsequent rise in the concentration of polymers induces precipitation to create a microspheric suspension (Kunchu et al., 2010)

1.11.6 Freeze Drying

Freeze-drying is effectively used in protein API microspheres preparation. The method is freezing, sublimation, main drying, and secondary drying. At the freezing step, account is taken of the eutectic point of the components.  During  the  process,  lyoprotectants  or cryoprotectants  will  stabilize  API  molecules  by removing  water,  creating  a  glass matrix,  lowering intermolecular  interaction  by  forming  hydrogen  bonds between the molecules or dipole - dipole interactions. It's a beneficial cycle for heat tolerant molecules, given its high expense. Freeze-drying produces solidification and then enables the reconstitution of particles in an aqueous media (Kunchu et al., 2010).

1.11.7 Single Emulsion Solvent Evaporation Technique

This process requires polymer dissolution in an organic solvent  accompanied  by emulsification  of  an  aqueous environment  containing  the  emulsifying  agent.  The resulting  emulsion  is  stirred  for  several  hours  in atmospheric conditions to allow the solvent to evaporate, which  is then  washed,  rinsed and  dried  in  desiccators. Designed and manufactured drugs microspheres with polymers by diffusion-evaporation method with emulsion solvent (Kunchu et al., 2010).

Figure 2: single emulsion technique

1.11.8 Double emulsification method

The Doppel-emulsion strategy requires mixing w / o / w or o / w / o processing the double emulsion. The aqueous solution of the product is distributed in a continuous lipophilic organic phase.  The continuous step which consists of a polymer solution eventually encapsulates medication Observed in the scattered aqueous layer to form primary emulsion.  Prior to introduction to the aqueous  solution  of alcohol to  form  primary emulsion, the pre-formed emulsion is subjected to homogenisation or  sonication.  The microspheres filled with the drug prolonged the  release  of the  medication  24  hours and were Observed to be diffusion and erosion regulated (Kunchu et al .,2010).

Figure 3: Double emulsion technique

1.11.9 Ionic gelation method

Ionotropic  gelation  is  depend  on  the  tendency  of polyelectrolytes  to  cross  connect  to  develop  hydrogel beads often called gelispheres in the existence of counter ions. Gelispheres  are  Circular  cross  linked  polymeric hydrophilic  agent  capable  of  substantial gelation  and thickening  in  model  biological fluids  and drug  release regulated  by  polymer relaxation  via  it.  The  hydrogel beads  are  formed by  dumping  a  drug-laden  polymeric solution  into  the  polyvalent  cations  aqueous  solution. The cations migrate through the drug-laden hydrophilic compounds, creating a three-dimensional lattice the moiety is ironically crosslinked. Biomolecules may also be placed into these gelispheres to maintain their three-dimensional form under moderate conditions (Kunchu et al., 2010).

1.13 Formulation of microspheres by Solvent Evaporation method

Microspheres containing Famotidine drug as a core material were prepared by Solvent Evaporation method. Drug (Famotidine), HPMC and EC were dissolved in a mixture of ethanol and dichloromethane (1:1) at room temperature (As in table 6). This was poured into 250 mL water containing 0.01% Tween-80 maintained at a temperature of 30–40 °C and subsequently stirred at 300 rpm agitation speed for 45 minutes to allow the volatile solvent to evaporate. The microspheres formed were filtered, washed with water and dried in oven at 37°C (Fartyal et al., 2011).

1.4 Evaluation parameter of drug loaded microsphere

1.4.1 Particle size

The particle size is one of the most important parameter for the characterization of microspheres. The size of microspheres was measured using Malvern Zeta sizer (Malvern Instruments). The dispersions were diluted with Millipore filtered water to an appropriate scattering intensity at 25°C and sample was placed in disposable sizing cuvette. The size data is documented in Table 13 (Singh and Vingkar 2008).

1.4.2 Zeta potential

The zeta potential was measured for the determination of the movement velocity of the particles in an electric field and the particle charge. In the present work, the microspheres was diluted 10 times with distilled water and analyzed by Zetasizer Malvern instruments. All samples were sonicated for 5-15 minutes before zeta potential measurements.  The zeta potential data is documented in Table 14 (?or?evi? et al., 2015).

1.4.3Quantitative analysis (Entrapment Efficiency)

%Entrapment efficiency was determined by indirect estimation. Drug -loaded microspheres were centrifuged at 15,000 rpm for 30 min using REMI Ultra Centrifuge. The non-entrapped drug (free drug) was determined in the supernatant solution using UV spectrophotometer.

The peak area was determined and amount of free drug is determined by extrapolating the calibration curve. And drug entrapment calculated by using below equation. The entrapment efficiency data is documented in Table 15 (Balla and Goli 2020).

Entrapment efficiency % = Total drug conc. - Supernatant drug conc. / total drug conc.*100

1.4.4 Scanning Electron Microscopic (SEM)

The electron beam from a scanning electron microscope was used to attain the morphological features of the drug loaded microspheres were coated with a thin layer (2–20 nm) of metal(s) such as gold, palladium, or platinum using a sputter coater under vacuum. The pretreated specimen was then bombarded with an electron beam and the interaction resulted in the formation of secondary electrons called auger electrons. From this interaction between the electron beam and the specimen’s atoms, only the electrons scattered at 90° were selected and further processed based on Rutherford and Kramer’s Law for acquiring the images of surface topography (Anwer et al., 2019).

1.5 In-vitro drug release study

The in-vitro drug release study of drug loaded Microsphere formulation was studied by dialysis bag diffusion method. Microspheres were dispersed into dialysis bag and the dialysis bag was then kept in a beaker containing 100 ml of pH 7.4 phosphate buffer. The beaker was placed over a magnetic stirrer and the temperature of the assembly was maintained at 37 ± 2 °C throughout the experiment. During the experiment rpm was maintained at 100 rpm. Samples (2 ml) were withdrawn at a definite time intervals and replaced with equal amounts of fresh pH 7.4 phosphate buffers. After suitable dilutions the samples were analyzed using UV–Visible  spectrophotometer at 304 nm. To analyze the in vitro drug release data various kinetic models were used to describe the release kinetics.To analyze the in vitro release data various kinetic models were use to describe the release kinetics. The zero order rate Eq. (2) describes the systems where the drug release rate is independent of its concentration. The first order Eq. (3) describes the release from system where release rate is concentration dependent. Higuchi described the release of drugs from insoluble matrix as a square root of time dependent process based on Fickian diffusion. The results of in vitro release profile obtained for all the formulations were plotted in modes of data treatment. 

1.6 Stability studies

The drug loaded Microsphere formulation was packed and were placed in the stability test chamber and subjected to stability studies at accelerated testing (25±2C and 60 ± 5% RH) and (40±2 0C and 70 ±5% RH) for 3 months. The formulation was checked for evaluation parameter particle size, zeta potential and Appearance at the interval of 30, 45, 60, 90 days (3 month) months. The formulation was tested for stability under accelerated storage condition for 3 months in accordance to International Conference on Harmonization (ICH) guidelines.

The formulation was analyzed for the change in evaluation parameter particle size, zeta potential and appearance. All Results were compared against final formulation of 0 days as the reference.

RESULTS AND DISCUSSION

2.1 Evaluation parameter of microsphere formulation

7.7.1 Particle size determination

Figure 6: Particle size (F3)

Figure 7: Particle size (F4)

Figure 8: Particle size (F5)

Table 6: Result of Particle size of all formulations

DISCUSSION  

The particle size is one of the most important parameter for the characterization of microsphere. The average particle sizes of the prepared microsphere formulation were measured using Malvern zeta sizer. Particle size analysis showed that the average particle size of microsperes was found to be range between 181.4 to 823.8 nm. These particle size values indicate that the all formulated microsphere is under the range of microsphere and F3 is the lowest particle size of all formulation shown in above table

Figure 9: Zeta potential (F1)

Figure 10: Zeta potential (F2)

Figure 11: Zeta potential (F3)

Figure 12: Zeta potential (F4)

Figure 13: Zeta potential (F5)

DISCUSSION

Zeta potential analysis is carried out to find the surface charge of the particles. The magnitude of zeta potential is predictive of the colloidal stability. Zeta potential was found to be all formulation range -0.5 to -44.7 mV with peak area of 100% intensity. These values indicate that the all formulated microsphere is stable. Results show in above table.

2.3 Entrapment efficacy

Table 8:   Entrapment efficacy

DISCUSSION

This might be due to the fact that the variation in entrapment efficiency was due to the changes in the polymer concentration. The prepared drug loaded microsphere formulation possesses high drug entrapment efficiency (90.03) of F3 formulation due to change in polymer concentration and all formulation EE % found to be in the range of 75.99 to 90.03%.

2.4 Scanning electron microscopy characterization of F3 formulation

DISCUSSION 

SEM analysis was performed to determine their microscopic characters (shape & morphology) of prepared microsphere. Drug loaded microsphere were prepared and dried well to remove the moisture content and images were taken using scanning electron microscopy. Scanning electron micrograph of the prepared microsphere at 50.00 kx magnification showed that the microsphere were smooth surface morphology and spherical shape. The smooth surface morphology and spherical shape of microsphere was clearly observed in the SEM images. Show in above figure 18.

       
           
       
    Figure 16: First order model of F3 formulation

Figure 17: Higuchi model of F3 formulation

       
           
       
    Figure 18: Korsmeyerpeppas

DISCUSSION

The data of percentage drug release formulation were shown in Figure. 19 to 22. For kinetic study following plots were made: cumulative   % drug release vs. time (zero order kinetic models); log cumulative % drug remaining vs time (first order kinetic model); cumulative   % drug release vs square root of time (Higuchi model); log cumulative % drug release vs log time (Korsmeyer–Peppas model). All Plots are shown in Figure. 19 to 22 and results are summarized in Table 17. Zero order kinetic models refer to the process of constant drug release from a drug delivery device independent of the concentration. The zero order graph of optimized formulation showed the constant drug release from the Formulation, the results of the zero order model was found to be  y = 8.776 x + 1.128 R  = 0.981. The first order kinetic model describes the release from system where release rate is concentration dependent. The results of first order kinetic model was found to be y = -0.142x+2.290 R?2; = 0.865. The Higuchi model is used to describe the limits for transport and drug release. The Higuchi model of formulation was found to be, y = 30.46x - 15.87 R?2; = 0.906. And the results of Korsmeyerpeppas kinetic model was found to be y = 1.693x + 0.394 R?2; = 0.880. In-vitro drug release was carried out using Franz diffusion cell. The In-vitro drug release data for F3 formulation was performed in pH phosphate buffer 7.4. The F3 formulation showed 95% drug release within 11 hrs. In the above table 14 R2 is correlation value. On the basis of best fit with the highest correlation (R2) value it is concluded that in the F3 formulation of microsphere follow the zero order kinetic model.