CONTROLLED DRUG DELIVERY FROM A NOVEL INJECTABLE IN SITU FORMED BIODEGRADABLE PLGA MICROSPHERE SYSTEM

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OBJECTIVES
The main intention of this research project were to achieve controlled drug delivery of micromolecules and macromolecules, such as proteins, from a novel injectable biodegradable poly(lactide-co-glycolide) (PLGA) microsphere system.
This system would overcome some of the disadvantages associated with the traditional methods for controlled drug delivery. On injection, the system would come in contact with water from aqueous buffer or physiological fluid and as a result, form solid matrix type microparticles entrapping the drug (in situ formed microspheres); the drug would be released from these microspheres in a controlled fashion.
The specific objectives of this research project were as follows: (1) To develop a novel method for controlled delivery of drugs from an in situ forming biodegradable PLGA microsphere system.
(2) To evaluate the effects of various formulation variables on the characteristics of this system. (3) To determine the effects of formulation, process and storage conditions on the reproducibility and stability of this system as well as the stability of the encapsulated proteins.
(4) To modify this novel microencapsualtion process, to produce in situ formed implant or isolated microspheres and also compare the characteristics of the three biodegradable devices: in situ formed implant v/s in situ formed microsphres v/s isolated microspheres.

INTRODUCTION
To avoid inconvenient surgical insertion of large implants. injectable biodegradable and biocompatible polymeric particles (microparticles and nanoparticles) could be employed for parenteral controlled-release dosage forms .
Microparticles of size less than 250 µm, ideally less than 125 ~Lrn are suitable for this purpose. Biodegradable polymers are natural or synthetic in origin and are decomposed in vivo, either enzymatically or non-enzymatically to produce biocompatible, toxicologically safe by-products which are further eliminated by normal metabolic pathways. Drugs formulated in polymeric devices are released either by diffusion through the polymer barrier, or by erosion of the polymer material, or by a combination of both diffusion and erosion mechanisms. The polymers selected for the parenteral administration must meet several requirements like biocompatibility, drug compatibility, suitable biodegradation kinetics and mechanical properties, and ease of processing.
Although a wide variety of natural and synthetic biodegradable polymers have been investigated for drug targeting or prolonged drug release, only a few of them are actually biocompatible. Natural biodegradable polymers like bovine serum albumin (BSA), human serum albumin (HSA), collagen, gelatin, and hemoglobin have been studied for drug delivery. The use of these natural polymers is limited due to their higher costs and questionable purity.
In the past two decades synthetic biodegradable polymers have been increasingly used to deliver drugs, since they are free from most of the problems associated with natural polymers. Poly(amides), poly( amino acids), poly(alkyl-cx.cyano acrylates), poly(esters), poly(orthoesters), poly(urethanes), and poly(acrylamides) have been used to prepare polymeric devices to deliver drugs.
poly(glycolide) (PGA), and especially the copolymer of lactide and glycolide referred to as poly(lactide-co-glycolide) (PLGA) have generated immense interest due to their excellent biocompatibility and biodegradability. Also PLGA has been approved by the U.S. FDA for a number of clinical applications including surgical sutures and as controlled-release microspheres. PLGA is shown to be biocompatible and degrades to toxicologically acceptable lactic and glycolic acids that are eventually eliminated from the body. Release of drugs from PLGA microspheres occurs by two mechanisms: (i) diffusion of the drug through a tortuous, water-filled path in the polymer matrix and (ii) matrix bioerosion (bulk hydrolytic degradation) after undergoing sufficient hydration. The actual release is a combination of both the processes.
There is a particular interest in controlled delivery of macromolecules like peptides and proteins through PLGA microspheres. Although a wide variety of pharmacologically useful peptide and protein based drugs have been recently developed by genetic engineering, their therapeutic use is restricted due to certain disadvantages: (i) on oral consumption they are subject to attack by the acidic and enzymatic environment in the stomach and the enzymes from the brush border membrane of the intestine, (ii) their high molecular weight and size impede their effective transport across the gastrointestinal membranes, and (iii) they have a short biological half-life and on injection they are quickly metabolized and eliminated. To achieve sustained blood levels of these drugs, minimize their denaturation or degradation, and to extend their biological half-life, their delivery by encapsulation in PLGA microspheres has become an interesting approach.
The literature on PLGA microspheres is full of different techniques describing their manufacture, where the microspheres are produced in a freeflowing, powder form. Some of the methods reported are : (i) single/double emulsification followed by solvent removal by evaporation or extraction, (ii) phase separation ( coacervation), and (iii) spray-drying. Most of these manufacturing processes suffer from drawbacks such as: (i) the microspheres need to be reconstituted (suspended) in an aqueous media, before they could be injected in the body, (ii) the hazards and environmental concern associated with the use of organic solvents like methylene chloride for the solubilization of PLGA polymer, and (iii) residual organic solvents remaining in the final microsphere product.
have described a novel implant system which is parenterally administered as a liquid and subsequently solidifies into a gel matrix (implant) in situ, from which the drug is released in a controlled manner. Although this implant system precludes the need for any surgery for its administration, it has a number of disadvantages: (i) the safety of solvents like N-methyl-2-pyrrolidone. used to formulate these systems is questionable and not well documented, (ii) the injection of these liquid implant systems and their subsequent solidification produce non-uniform matrix implants having variable consistency and geometry.
and (iii) due to formation of matrix implants having inconsistent texture. shape and size, the drug release from them is variable and unpredictable.
The present process of microsphere formation is based on the principle of coacervation. This method overcomes the problems faced by the above systems by forming a dispersion of PLGA micro globules ("premicrospheres" or "embryonic microspheres") in an acceptable vehicle mixture (continuous phase) and whose integrity is maintained by use of appropriate stabilizers. Of serious concern are the problems associated with the oral administration of If a drug cannot be administered orally due to any of the above reasons, a parenteral route of delivery is an alternative. One advantage that a parenteral controlled release dosage form has over oral controlled release dosage forms is patient compliance (2). Although an oral dosage form might have a good bioavailability, a long-acting parenteral dosage form that is safe and efficacious for days or weeks or months could be beneficial because it ensures that the patient is receiving medication. Also a parenteral controlled release dosage form is preferred over conventional parenteral dosage form for chronic treatment where routine multiple injections could be inconvenient and painful. Parenteral controlled release dosage forms are also effective in site-specific drug delivery.
thereby improving its efficacy and reducing its toxicity. The main disadvantage of these dosage forms is that once administered, they cannot be easily removed (2). This could be a problem for the patient if a drug was no longer needed. or worse if it caused an undesirable reaction. Although a wide variety of natural and synthetic biodegradable polymers have been investigated for drug targeting or prolonged drug release, only a few of them are actually biocompatible. Natural biodegradable polymers like bovine serum albumin (BSA), human serum albumin (HSA), collagen, gelatin, and hemoglobin have been studied for drug delivery (1 ). The use of these natural polymers is limited due to their higher costs and questionable purity ( 1 ).
This review provides a comprehensive outlook on different techniques of preparation of various drug loaded PLGA devices, with special emphasis on preparing microparticles. Certain issues about other related biodegradable polyesters like PLA and PGA have been discussed as well.

HISTORICAL DEVELOPMENT OF DRUG DELIVERY USING PLGA
The discovery and the synthetic work on low molecular weight oligomeric forms of lactide and/or glycolide polymers was first carried out several decades back (3, 5). The methods to synthesize high molecular weights of these polymers were first reported by Lowe (3).
During the late 1960s and early 1970s a number of groups had published pioneering work on the the utility of these polymers to make sutures/fibers (2, 3, 5, 12). These fibers had several advantages such as good mechanical properties.
The biodegradation, biocompatibility, and tissue reaction of PLA and PLGA have been extensively investigated and well documented by many researchers (5, 14). The first work on parenteral controlled release of drugs using PLA was reported by Boswell, Yalies, Sinclair, Wise,and Beck (3,5) . Since then an ocean of literature on drug delivery using PLA, and especially PLGA has been published. Various polymeric devices like microspheres, microcapsules, nanoparticles, pellets, implants, and films have been fabricated using these polymers for the delivery of a variety of drug classes.

SYNTHESIS OF PLGA COPOLYMER
Low molecular weight PLGA can be prepared by direct condensation (polyesterification) of lactic and/or glycolic acids (5, 12). Temperatures as high as 130-190° C are required for the condensation process and the water generated is removed by boiling, using vacuum, purging with nitrogen, or azeotropic distillation with an organic solvent (3, 12). An acid catalyst like antimony oxide increases the reaction rate if used at reaction temperatures below 120° C. but above this temperature water removal is the rate-limiting step (3,12). This method yields PLGA having molecular weight of -l 0,000 ( 12). The low molecular weight PLGA has limited biomedical application, due to its poor mechanical strength and faster degradation (3).
Intermediate and high molecular weight PLGA (-10,000-40,000) can be prepared by the ring-opening polymerization of the cyclic dimers (cyclic diester of lactic and/or glycolic acids) as the starting materials (3,5,12,14). The advantage of this method is that no water removal/dehydration method is needed in the polymerization system (3). Also the cyclized monomer(s) and the linear form of the polymers produced can be readily purified (3). Compounds of lead.
tin, cadmium, zinc, antimony, and titanium have been used as catalyst to initiate the polymerization process (12,14). Acid catalyzed bulk polymerization (melt method) for two to six hours at around 175° C is generally employed for preparation of PLGA from lactide and glycolide monomers (3 ). The molecular weight of the resultant PLGA is determined by the concentration of the catalyst added (12). Monomer purity of99.9% or greater and monomer acidity of 0.05% or less are required with the starting lactide and glycolide materials (5). Also important are the low levels of humidity in the processing area (5).

PHYSICAL, CHEMICAL, AND BIOLOGICAL PROPERTIES OF PLGA
It is important to understand the physical, chemical, and biological properties of the polymer before formulating a controlled drug delivery device .
The various properties of the polymer and the encapsulated drug directly ( influence other factors like the selection of the microencapsulation process, drug release from the polymer device, etc. (1).
PLA can exist as the optically active stereoregular polymer (L-PLA) and a optically inactive racemic polymer (D, L-PLA) (1 , 5, 9). L-PLA is found to be semicrystalline in nature due to high regularity of its polymer chain while D, L-PLA is an amorphous polymer because of irregularities in its polymer chain structure (3 , 9). Hence the use of D, L-PLA is preferred over L-PLA as it enables more homogeneous dispersion of the drug in the polymer matrix (9. 13 ). PGA is highly crystalline because it lacks the methyl side groups of the PLA (3, 9).
Lactic acid is more hydrophobic than glycolic acid and hence lactide-rich PLGA copolymers are less hydrophilic, absorb less water, and subsequently degrade more slowly (1, 3, 13).
The molecular weight and polydispersity index of the polymer are factors which affect the mechanical strength of the polymer and its ability to be formulated as a drug delivery device (3, 5, 12). Also these properties may control the polymer biodegradation rate and hydrolysis (3, 12). The commercially available PLGA polymers are usually char!lcterized in terms of intrinsic viscosity which is directly related to their molecular weights (3).
The degree of crystallinity of the PLGA polymer directly influences its mechanical strength, swelling behavior, capacity to undergo hydrolysis, and subsequently its biodegradation rate (3) . The resultant crystallinity of the PLGA copolymer is dependent on the type and the molar ratio of the individual monomer components (lactide and glycolide) in the copolymer chain (1). PLGA polymers containing 50:50 ratio of lactic and glycolic acids are hydrolyzed much faster than those containing higher proportion of either of the two monomers (5, 12). PLGAs prepared from L-PLA and PGA are crystalline copolymers while those from 0, L-PLA and PGA are amorphous in nature (3, 5). Gilding and Reed have pointed out that PLGAs containing less than 70 % glycolide are amorphous in nature ( 18).
The degree of crystallinity and the melting point of the polymers are directly related to the molecular weight of the polymer (3, 5).

(
The glass transition temperature (Tg) of the PLGA copolymers are above the physiological temperature of 37° C and hence they are glassy in nature (3. 5).
Thus they have a fairly rigid chain structure which gives them significant mechanical strength to be formulated as drug delivery devices ( The carboxylic end groups present in the PLGA chains increase in number during the biodegradation process as the individual polymer chains are cleaved; these are known to catalyze the biodegradation process (3, 5). The biodegradation rate of the PLGA copolymers are dependent on the molar ratio of the lactic and glycolic acids in the polymer chain, molecular weight of the polymer, the degree of crystallinity, and the Tg of the polymer (3, 5, 13). A three phase mechanism for the PLGA biodegradation has been proposed (21 ): 1. Random chain scission process. The molecular weight of the polymer decreases significantly, but no appreciable weight loss and no soluble monomer products formed.
2. In the middle phase a decrease in molecular weight accompanied by rapid loss of mass and soluble oligomeric and monomer products are formed. The PLGA polymer biodegrades into lactic and glycolic acids ( 1-3, 5, 12.

13). Lactic acid enters the tricarboxylic acid cycle and is metabolized and
subsequently eliminated from the body as carbon dioxide and water (1-3 , 5. 9). ln a study conducted using 14 C-labeled PLA implant, it was concluded that lactic acid is eliminated through respiration as carbon dioxide (22). Glycolic acid is either excreted unchanged in the kidney or it enters the tricarboxylic acid cycle and is eventually eliminated as carbon dioxide and water (3).

METHODS OF PREPARING VARIOUS PLGA DEVICES [1] MICROPARTICLES
A number of microencapsulation techniques have been developed and reported to date. The choice of the technique depends on the nature of the polymer, the drug, the intended use, and the duration of the therapy (1 , 2, 4, 5, 10). The microencapsulation method employed must include the following requirements (1, 2, 23): (i) The stability and biological activity of the drug should not be adversely affected during the encapsulation process or in the final microsphere product.
(ii) The yield of the microspheres having the required size range (upto 250 µm, ideally < 125 µm) and the drug encapsulation efficiency should be high.
(iii) The microsphere quality and the drug release profile should be reproducible within specified limits.
The microspheres should be produced as a free flowing powder and should not exhibit aggregation or adherence.

A. Solvent Evaporation and Solvent Extraction Process (1) Single emulsion process
This is essentially an oil-in-water (o/w) emulsion process. The polymer is first dissolved in a water immiscible, volatile organic solvent; dichloromethane (DCM) most commonly used. The drug is then added to the polymer solution to produce a solution or dispersion of the drug particles (particle size of the drug added to be< 20 µm) (4). This polymer-solvent-drug solution/dispersion is then emulsified (with appropriate stirring and temperature conditions) in a larger volume of water in presence of an emulsifier (such as poly (vinyl alcohol) (PY A)) to yield an o/w emulsion. The emulsion is then subjected to solvent removal by either evaporation or extraction process to harden the oil droplets (I 0). In the former case the emulsion is maintained at reduced pressure or at atmospheric pressure and the stirring rate reduced to enable the volatile solvent to evaporate ( 4, 10). In the latter case the emulsion is transferred to a large quantity of water (with or without surfactant) or other quench medium, into which the solvent associated with the oil droplets diffuses ( 4, 10). The solid microspheres so obtained are then washed and collected by filtration, sieving, or centrifugation ( 4 ). These are then dried under appropriate conditions or are lyophilized to give the final free flowing injectable microsphere product.
It should be noted that the solvent evaporation process in a way is similar to the extraction method, in the sense that the solvent must first diffuse out into the external aqueous dispersion medium before it could be removed from the system by evaporation ( 4, 10 and water are used as dispersed and continuous phases respectively. DCM is widely used because it is a good solvent for the polymers and due to its high volatility it can be easily removed by evaporation. A major problem with the use of DCM is its potential toxicity (28).
Chlorinated solvents in general are considered hazardous to environment and undesirable for use in manufacturing processes (28) ( 4 7). In the former case, most of the acetone was first al lowed to diffuse out from the dispersed organic phase (chloroform-acetone mixture) into the external aqueous phase, followed by gradual evaporation of the residual solvents to give the final microspheres. In the latter case, the o/w emulsion was first subjected to solvent (DCM) evaporation for a certain period until semisolid droplets were obtained and the residual DCM was removed by the extraction process in a large volume of water. Microspheres from evaporation-extraction process were less porous and exhibited better encapsulation than those prepared from extraction-evaporation process ( 4 7). Extraction process using lidocaine base resulted in encapsulation efficiency of less than l 0% (33). The same group also reported a better product from the extraction process for the drug ketoprofen in terms of drug content, loading efficiency, particle size, and surface feature as against the evaporation process ( (

2) Double (multiple) emulsion process
The double emulsion process is essentially an water-in-oil-in-water (w/o/w) method and is best suited to encapsulated water-soluble drugs like  causing reduction of the microsphere size and increase in particle porosity due to better stabilization of the inner w/o emulsion (100). Other researchers have also reported use of L-a-phosphatidylcholine ( 112). In another study it was found that a decrease in the DCM phase volume yielded particles with dense core (81 ).
The entrapment efficiency of the drug increased with decrease in drug loading and increase in particle size (84). However other groups have found no Cohen et al. have reported that for microspheres in which the inner emulsion was prepared using low shear (e.g. vortex mixing), the particles were large in size and the drug encapsulation was low as compared to microspheres in which the inner emulsion was prepared using high shear (e.g. probe sonication) which yielded smaller particles with higher encapsulation efficiency (81 ).
However Sah et al. reported no effect of the shear rate (to prepare the o/w emulsion) on the encapsulation efficiency and the final particle size of PLA/PLGA microcapsules; particles prepared from low shear rate were however more porous than those prepared from high shear rate (116).  and i.m. routes (85 , 86, 88) and a three-month release profile following a s.c.
injection (87) has been reported by these researchers.

B. Phase Separation (Coacervation)
The The phase separation method, unlike the o/w emulsification method is suitable to encapsulate both. water-soluble as well as water-insoluble drugs. since its a non-aqueous method. However the coacervation process is mainly used to encapsulate water soluble drugs like peptides, proteins, and vaccines. The addition rate of first nonsolvent should be such that the polymer solvent is extracted slowly, so that the polymer has sufficient time to deposit and coat evenly on the drug particle surface during the coacervation process (4) . The concentration of the polymer used is important as well, since too higher concentrations would result in rapid phase separation and nonuniform coating of the polymer on the drug particles. Due to absence of any emulsion stabilizer in the coacervation process, agglomeration is a frequent problem in this method ( 4 ).
The coacervate droplets are extremely sticky and adhere to each other before the complete phase separation or the hardening stages of this method. Adjusting the stirring rate, temperature, or the addition of an additive is known to rectify this problem (4).
Unlike the solvent evaporation/extraction process, the requirement of solvents for the polymer are less stringent since the solvent need not be immiscible with water and the boiling point can be higher than that of water ( 4 ).

CONCLUSION AND FINAL REMARKS
The salient features of the novel microencapsulation process and the drug delivery sytem described in this .research project are as follows: (1) The sytem excluded the use of unacceptable organic solvents like methylene chloride and used acceptable vehicle mixture instead to prepare biodegradable PLGA microspheres.
(2) The sytem formed drug containing PLGA microglobules ("premicrospheres" or "embryonic microspheres") which could be considered as precursors to the final microsphere product: these on coming in contact with water hardened to form discreet PLGA microspheres (in situ formed microspheres) which subsequently exhibited non-variable.
predictable, and controlled drug release profile.
(3) Unlike the traditional methods, this system precluded the need for reconstitution of the PLGA microspheres as they are formed in situ.
( 4) Various formulation varibles affected the characteristics of this system.
The formulation and process conditions did not adversely affect the physical stability of the encapsulated protein drugs.
(6) Besides in situ forming microspheres, the novel microencapsulation method can be modified to produce in situ formed implant or isolated microspheres; these drug loaded devices exhibited different characteristics.
This research project makes a significant overall contribution to the knowledge of the underlying theoretical principles of drug delivery through biodegradable devices and in particular, problems associated with protein drug delivery.
(8) The novel nature of the system provides a high probability that a patent application would be filed.