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Anisotropic surface coatings controls drug delivery from reservoir devices similar to that of erodible systems.

Introduction

The lipid composition of the cell membranes is diverse across the membrane, and that produce anisotropic lateral condensation of these lipids to condensed- regions and fluid-regions [1]. This anisotropic condensation of lipids play an important role in improving the functional efficiency of natural plasma membranes in terms of, tailor made signal presentation [2], altered membrane elasticity [3], resealability [4], and other mechanical properties of the membrane. These factors can play an important role in the delivery of agents from cells [4]. However, these properties are rarely explored for engineering material and device properties.

Many studies have been done in the past to develop biomimetic laterally self-assembling synthetic systems using lipids [5-7] and polymers [8, 9]. The coatings made of self-assembly are simple in terms of processability yet versatile in terms of its functional capacity rendering it as one of the most sort-after technique for the surface modification of the futuristic micro and nanotechnology devices [10-12]. We have recently demonstrated that, on model lipid membranes, the membrane composition can be varied to modulate the size of cholesterol rich domains, which can intern modulate its lateral mechanical properties to control the macrophage adhesion responses [13]. These anisotropy dependent changes in lateral mechanical properties can be dynamically modulated by physical parameters [14] such as membrane curvature [4], polarity of supported substrate as well as even to the diffusion flux across the membranes [15]. For example, if anisotropic surface coatings can influence the diffusion flux from a delivery device, it can be explored for controlling the drug delivery across the membrane.

Drug delivery systems that can produce zero-order release kinetics are useful for prolonged local drug delivery of agents [16, 17]. Such systems show reduced burst release and prolonged controlled delivery from reservoir to the sink in a concentration independent manner, depending upon the degradation of the polymer [18]. Erodible polymers are widely being used for the said purpose [1920]. Newer systems and mechanisms to achieve concentration independent prolonged controlled delivery are on a constant search [21]. Strategies to modify the conventional diffusion based easily adaptable drug delivery system that can produce reduced burst release and prolonged controlled delivery in a concentration independent manner will be of great interest for new nanotechnological systems [22, 23]. We hypothesize that anisotropic lipid coatings can produce concentration-independent reduced burst release and prolonged controlled drug delivery from reservoir to sink.

In this work, we have developed a chitosan (CHIT) based porous microsphere for loading the drug molecules. We have used insulin as a model drug to load into the CHIT microspheres to prepare the CHITIns. The microspheres were further coated with model lipid coatings. Basically three lipid coatings are used for coating the CHITIns, which include (a) (PC) phosphatidylcholine to prepare PCCHITIns (b) (3L) with the mixed lipid composition, phosphatidylcholine: cholesterol (Chol): galactocerebroside (GalC) to prepare (3LCHITIns), and (c) (4L) with the mixed lipid composition, phosphatidylcholine: cholesterol: galactocerebroside: phosphatidylethanolamine (PE) to prepare (4LCHITIns). The lipid composition of 3L [5] and 4L [13] was optimized in our earlier studies. These devices were characterized by different methods and evaluated for insulin delivery. Our results show that, anisotropic lipid coatings can control the drug delivery in a concentration independent manner from the device. The delivery profile can be fine tuned depending upon the composition of the coating lipid membrane.

Materials and Methods

L-a-Phosphatidylcholine (egg yolk) (PC), L-aPhosphatidylethanolamine (PE) and Galactocerebrosides (GalC) (from bovine brain) were purchased from Sigma Chemicals Co. St Louis MO, USA. Cholesterol (Chol) was purchased from (Himedia Pvt. Ltd, Bombay), Chitosan (CHIT) of (Approx. M.Wt. 2.70 kDa) (85% deacetylated) (CIFT, India) (was used as the polymer substrate in these studies.

Preparation of lipid coating solutions

Stock solutions of lipids was freshly prepared using a known volume of spreading solvent mixture, Cyclohexane/ n-Butanol (CH/n-but) (5:2 v/v) to that the preweighed lipids were dissolved. The lipid concentrations had been expressed in ratios (w/w), with respect to the concentration of the PC. The working solutions were prepared by mixing appropriate volumes from stock solutions. The composition of the (3L) lipid solution with the mixed lipid composition, (PC: Chol: GalC) (1: 0.35: 0.125) w/w and the one with incorporation of PE as (4L) lipid solution with the mixed lipid composition, (PC: Chol: GalC: PE) (1: 0.35: 0.125: 0.29) w/w was prepared as the working solutions. All working lipid mixtures were produced immediately prior to use.

Preparation of porous CHIT microspheres

The CHIT microspheres were prepared by using a CHIT 2% (w/v) in 1.5% v/v acetic acid solution. The CHIT solution (25ml) was added drop wise into 200ml of ethanolic NaOH (10% (w/v) solution with the help of a syringe with 15G needle in 250ml beaker under stirring (@ 2000rpm). The stirring was continued for next 2hrs. The resulting microspheres were washed with copious amount of water to remove the alkali until the pH comes to 5.6. These microspheres were swollen at pH 5.5 neutralised phthalate buffer. Again the microspheres are washed with copious amount of deionised water. They were sequentially introduced into acetone/water mixture (10/ 90, 30/70, 50/50, 70/30, 100/0) v/v and freeze dried (Labconco) after removing the excess acetone, to get a porous scaffold.

Preparation of drug delivery device

The drug delivery device was prepared by loading the model drug insulin followed by the lipids into the porous CHIT microspheres. For that, the required amount of insulin was added to these microspheres and freeze dried for diffusion filling into the microspheres. The incorporation of the lipid into the microspheres was done, after precipitating the entrapped drug with acetone. A quantity equivalent to 100mg of microspheres were weighed to a tarred beaker to that 5ml of the lipid solution was added and allowed to dry. A solution of 50mg% of PC or lipid mixture in cyclohexane/ n-butanol (CH/ n- but) (5:2) v/v solution was added into the microspheres. Thus formed delivery device was dried completely in a vacuum oven, sealed aseptically into bottles and stored at 4[degrees]C until used for further studies.

Microscopy of the microspheres

Stereo Zoom Microscopy: The stereo microscopy of the microspheres was done by using stereo zoom microscope (Leica MZ 6) at 100x magnification. Scanning electron microscopy (SEM): The surface or internal morphology of CHIT microspheres were examined by SEM. The surface morphology as well as the internal structure (cut open) of the CHIT was studied after mounting on an aluminium using SEM after gold coating. Dehydration of the tissue specimens were carried out with ethanol in a graded series to avoid shrinkage. For dehydration the samples were dipped for 15min each in a series of 30. 50. 70. 80 90, 95 & 100% ethanol. Finally the samples were critical point dried (CPD) in liquid C[O.sub.2]. The samples were mounted onto aluminium stubs using double sided adhesive tape, further coated with gold film and were observed under SEM (Model S-2400, Hitachi, Japan)

Pore volume measurements

The pore volume of the microspheres was evaluated by measuring the volume occupied by the organic solvent. For that the swollen microspheres were exchanged with acetone/water mixture (10/90, 30/70, 50/50, 70/30, 100/0) v/ v as explained earlier, Then with acetone/ dichloromethane (50/50) v/v mixture and finally with dichloromethane alone. The volume occupied by the DCM was evaluated gravimetrically after centrifugation at 10,000rpm for 10 min in a sealed test tube.

Drug release studies

Insulin (Human 40 IU/ ml) was used as a model drug in these studies. The drug was loaded into the microspheres by diffusion filling method. Drug content and drug release studies of the microspheres was done using Lowry method using the Folin-Cu reagent. Briefly, 100mg of the microspheres dispersed in 1ml of the buffer is tied into a dialysis bag tubing (M. Wt. cutoff 33,000) and was incubated in 19 ml (total dissolution medium = 20ml) of the PBS (7.4). A quantity equivalent to 200ml of the samples were taken at specified intervals (replaced with the buffer) and analyzed by Lowry method using UV-Visible spectrophotometer (Model UV 160A) at 750 nm.

The percentage drug released was quantified using the following formula,

Drug Release (%) = (Released insulin/ Total insulin) X 100.

Statistical analysis

Independent experiments were done in all the studies and the data is represented as mean [+ or -] standard deviation (SD).

Results and Discussion

In this work chitosan (CHIT) based microspheres were used as a model delivery device to represent the reservoir based delivery system. The insulin was used as a model macromolecular hydrophilic drug, because its release properties were unaffected by the device properties. Such large microsphere based porous device was developed to demonstrate the exclusive capacity of the lipid coatings to control the release properties. Where the lipid coatings not only fill the cavities but also coat the surface of the delivery device, thus produce multiple barriers for the drug to diffuse from the reservoir to the sink.

The figure 1 shows the CHIT microsphere used in these studies. The specific reservoir characteristics of the microspheres are compiled in the table-1. The figure-1a shows that, the microspheres have a dimension upto 3mm, and which provides lower surface area with respect to the reservoir volume (H" 4ml/gm). The surface topography of the microspheres by SEM (figure 1b(i)) shows that, the outer thin membrane is continuous throughout the microspheres with intermittent small pores on its surface. The figure 1b(ii) below shows that, the internal structure of the microspheres is highly porous with large pores, small pores and inter-connected channels, starting less than 200pm in the case of large pores, reducing at the rate of one order magnitude in smaller pores and interconnected channels. As compared to the size of the molecules these dimensions are at least 4 to 5 order magnitude higher than the size of the model drug, enable free diffusion from reservoir to sink. These microspheres are uniform spherical microspheres, with porous interconnected cavities, thin walls and a continuous outer membrane which can hold large amount of liquid medium into its reservoir. This helps in creating a constant flux for diffusion of the agent from the reservoir to the sink. The thin outer membrane acts as rate control membrane between the reservoir and the sink producing limited control over the rate of delivery of agents from reservoir to the sink.

The said microspheres are loaded with insulin to prepare CHITIns and coated with the lipids. For that, the microspheres are loaded with insulin and the surface is modified with PC for PCCHITIns, 3L for 3LCHITIns and 4L for 4LCHITIns. Further the release studies are performed by In vitro studies in PB 7.4 at ambient conditions. The figure 2 shows the drug release from the PCCHITIns. In control (figure 2a), CHIT microspheres loaded with insulin (CHITIns), a burst release upto 50% is observed within first 2h. A controlled release upto 4th day is observed, wherein maximum release up to 60% is observed (figure2a). The studies are continued for 2 months and during the period up to 80% release are observed (figure 2a). In the case of PC coated CHITIns (PCCHITIns) microspheres, the PC forms an isotropic lipid membrane that alone has reduced the burst release from the reservoir type system and produced a more controlled drug delivery (figure 2b). In the case of PCCHITIns at 2h 20% release is observed, and at 4th day upto 45% and in two months release upto 80% is attained (figure 2a).

Subsequently, the CHITIns matrix is modified with 3L (3LCHITIns) and 4L (4LCHITIns) coatings to check whether the anisotropic lipid coatings have an influence on the drug release profile. The figure 3 shows the insulin release from the modified matrices. As compared to control CHITIns (figure 3a), In both the 3LCHITIns (figure 3b) and 4LCHITIns (figure 3c) less than 10% insulin is released in 2h against 60% released in its control matrix, and in 4days 30% is released in 3LCHITIns and 4LCHITIns against 80% in its control matrix. By 2months only up to 60% drug is released from 3LCHITIns and 4LCHITIns microspheres. The trend shows that, the release is continuing for further period of time.

The aim of the work is to device a strategy to produce a concentration independent drug release from reservoir type diffusion controlled drug delivery systems. In reservoir type systems the free diffusion of the drug molecules from the reservoir to the sink produce concentration dependent release profile (first order). To overcome this limitation we explored the capacity of the lipid layers to form cubic phases [24], assuming under confinement such cubic phase like period systems can form multiple gates for the drug to diffuse from the reservoir to the exterior. Our results show that, lipid systems under confinement in cavities are capable of producing a concentration independent release profile (Scheme 1). Further, anisotropic lipid systems are further efficient to control the release profile. In addition, we have also demonstrated that the anisotropy can be modulated to control the release profile. This promising results warrant further detailed analysis of the delivery system with respect to specific application.

Conclusion

We have developed a cell-mimetic controlled delivery device, wherein composition dependent membrane dynamics is predominantly controlling the delivery of the model drug insulin from reservoir to sink in a concentration independent manner. For that, we have prepared large sized porous chitosan microspheres with high internal volume and loaded with the model drug insulin, which is coated with either isotropic PC lipid layer or anisotropic mixed lipid layers. The release of insulin was concentration dependent and the release appeared steady and continuous in the case of chitosan matrix, giving a burst release above 50% in first 2h and controlled delivery of next 10% is observed at 4th day, while 60% of drug goat released. In the case of isotropic PC coater matrix the burst release has reduced to 20% and controlled delivery was observed up to 15days. In the case of anisotropic lipid coated matrix the burst release has reduced to 5% against 60% in control and controlled delivery up to 2 months to reach 80% is observed. Overall the anisotropic lipid mixture based coatings reduced the burst release to less than 10% at 2h against the 60% release in its control. At 4days upto 30% release is observed against the 80% release in control. By 2months only upto 60% insulin is released from the anisotropic lipid coated microspheres. Between the anisotropic lipid layers, phosphatidylethanolamine incorporated lipid membrane, one with domain size less than 10nm, has shown enhanced release at later time points. The lipid composition depended changes in the drug release shows that it's a tunable drug delivery system where membrane dynamics regulate the drug delivery profile. The control achieved by these coatings on reservoir type system warrants further detailed analysis to understand the mechanism of release profile.

Acknowledgement

We express our sincere thanks to the Director SCTIMST, Head BMT Wing, SCTIMST for providing the facilities. Dr. Kaladhar Kamalasanan expresses his sincere thanks to the SCTIMST for Chitra High Value Fellowship for his personal support.

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K. Kaladhar, Chandra P. Sharma

Biosurface Technology Division, Biomedical T echnology Wing, Sree Chitra Tirunal Institute for Medical Science and Technology, Poojappura, Thiruvananthapuram, Kerala, India.

* Corresponding authors, Prof. C.P. Sharma and Dr. Kaladhar, drcpsharma@rediffmail.com

Received 21 November 2014; Accepted 5 December 2014; Available online 10 December 2014

Table 1: The specific reservoir characteristics
of the microspheres

Structure of CHIT microspheres

Size                             3.18+/-0.12 mm

Weight                           0.2914+/-0.016 mg

Pore volume                      3.89+/-0.018 ml/gm
  (as per the procedure 2.2.6)

Internal Architecture

Larger Pore Dia                  188-200 [micro]m

Small Pore Dia                   80-120 [micro]m

Inter Connected Channels         40-55 [micro]m

Bridges                          2.5 [micro]m

Outer Film Thickness             2-5 [micro]m
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Title Annotation:Original Article
Author:Kaladhar, K.; Sharma, Chandra P.
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Oct 1, 2014
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