âLiquid Crystalline Nanoparticlesâ: Rationally Designed Vehicle To

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“Liquid Crystalline Nanoparticles”: Rationally designed vehicle to improve stability and therapeutic efficacy of insulin following oral administration Ashish Kumar Agrawal, Kuldeep Kumar, Nitin Kumar Swarnakar, Varun Kushwah, and Sanyog Jain Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on May 1, 2017

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Molecular Pharmaceutics

“Liquid Crystalline Nanoparticles”: Rationally designed vehicle to improve stability and therapeutic efficacy of insulin following oral administration Ashish Kumar Agrawalψ,1,2,*, Kuldeep Kumarψ,1, Nitin Kumar Swarnakar1,3,Varun Kushwah1, Sanyog Jain1,* ψ 1

Authors contributed equally

Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics,

National Institute of Pharmaceutical Education and Research, SAS Nagar, Punjab – 160062, India. 2

Current affiliation: James Graham Brown Cancer Center, University of Louisville, Louisville,

KY 40202, USA. 3

Current affiliation: Graduate College of Biomedical Sciences, Western University of Health

Sciences, Pomona, California 91766-1854, USA.

* To whom correspondence should be addressed: E-mail: [email protected]; [email protected] Telephone: Tel.: +91-172-2292055, Fax: +91-172-2214692, Tel:+1-502-852-3684, Fax: +1-502852-3842

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract In the present report we investigate the feasibility of liquid crystalline nanoparticles (LCNPs) to improve the stability and therapeutic efficacy of insulin following oral administration. Compatibility studies of different formulation ingredients with insulin and extensive optimization of different process variables resulted into the formation of LCNPs with particle size of 245.50 ± 6.36 nm, PDI 0.220 ± 0.042, zeta potential -18.30 ± 1.27 mV with an entrapment efficiency of 44.17 ± 1.47%. Mannitol (5% w/v) was identified as suitable cryoprotectant to produce freeze dried LCNPs without affecting the critical quality attributes of the LCNPs. LCNPs demonstrated excellent stability in simulated biological fluids by simultaneously retaining the chemical and conformational stability of the insulin entrapped within the LCNPs. A sustained release of insulin was observed for up to 24 h in PBS (pH 7.4). Developed LCNPs demonstrated remarkably higher caco-2 cell uptake in comparison with free Insulin-FITC and more than double the cumulative hypoglycemia in comparison with subcutaneously administered insulin solution in diabetic rats. Data in hand suggests that the proposed formulation strategy can be exploited for improving the therapeutic efficacy of the biomacromolecules like insulin.

Keywords: Liquid crystalline nanoparticles; insulin; oral delivery; caco-2 cell; glucose


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1.

Introduction

Diabetes mellitus is a group of metabolic diseases, typically characterized by high blood glucose level either due to insufficient amount of insulin produced in the body or due to the lack of response produced by cells against insulin.1 Although many classes of antidiabetics have been approved by FDA yet, insulin is needed for efficient management of diabetes at certain stages and considered as a gold standard therapy to manage the blood glucose level.2 Normal physiological pattern of insulin secretion involves the availability of majority of the insulin directly to the liver which is considered as primary organ involved in the glucose homeostatis. The classical subcutaneous administration of insulin is quite different from the natural physiological pattern which makes the insulin first available peripherally which results into transient hypoglycemia, peripheral hyperinsulinaema, lipoatrophy, lipohypertrophy and obesity.3 Moreover, this mode of administration can cause psychological stress leading to poor patient compliance. Oral delivery of insulin can overcome the drawbacks associated with conventional therapy however, being a peptide, when administered orally, insulin is prone to enzymatic and gastric degradation which is the major hurdle in developing oral dosage form of insulin. Moreover, higher molecular weight of the insulin does not allow it to permeate through the gastro intestinal epithelium.4, 5 The challenges of administering insulin orally have been addressed over the last several decades with a view of helping millions of diabetic patients from the pain and distress caused due to insulin injections. Several approaches6, which include colon delivery of insulin7, use of methaacrylic acid based hydrogel8, nanoemulsion coated with alginate/chitosan9, pH sensitive chitosan/alginate

nanoparticles10,

Hydroxyethyl-Aspartamide

penetratin

Copolymers

base

derivative-based

nanoaggregates12,


nanocomplexes11,

salecan-based

Poly-

pH-Sensitive


hydrogels13, Calcium phosphate-PEG-insulin-casein particles14, intestinal patches15, liposomal formulations4, have been reported to overcome the challenges following oral administration. Most of them were found to have issues related to low bioavailability, sub optimal efficacy of insulin or the problems related to industrial scale up. In the present work the feasibility of “Liquid Crystalline Nanoparticles” (LCNPs) has been tested for improved cellular uptake and therapeutic efficacy. LCNPs are the well-defined, thermodynamically stable, self-assembled lipid structures formed when these lipids are exposed to polar water phase. Insulin is supposed to be protected in the hydrophilic channels formed during the formation of LCNPs and to be bioavailable due to the variety of uptake mechanisms followed by nanoparticles. 2.

Methods

2.1. Materials 3,7,11,15-tetramethyl-1,2,3-hexadecanetriol (Phytantriol) and different grades of Pluronic® (F127, F-68 and F-87) were provided as generous gift from Danisco Pvt. Ltd, India, DSM Nutritional Products, Inc., Germany and BASF, Germany, respectively. Insulin (recombinant human), streptozotocin, acrylamide, N,N'-Methylenebisacrylamide, ammonium persulpahte (APS), tetramethylethylenediamine (TEMED), Insulin-FITC (>1 mole of FITC in 1 mol of insulin) were purchased from Sigma Aldrich, St. Louis, MO. Trifluoroacetic acid, pancreatin and pepsin were procured from Loba Chemie (Mumbai, India). Ultrapure water (LaboStar™ ultrapure water systems, Germany) was prepared in house and used in all the experiments. All other reagents were of analytical grade.


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2.2. Preliminary compatibility studies of insulin with different formulation ingredients Preliminary screening of different formulation ingredients was performed based on compatibility of insulin with different formulation ingredients and their effect on insulin conformation was evaluated by using circular dichroism (CD) spectrophotometer (Jasco J-815). 2.2.1. Solvents Insulin solution (150 µg/ml) was mixed with equal volume of ethanol and THF and kept for stirring at 1200 rpm. Samples were collected after 24 h and analyzed by using Jasco J-815 CD spectrometer. 2.2.2. Surfactants Pluronic F-127, Pluronic F-87 and Pluronic F-68 (25 mg each) were added separately to insulin solution (5 ml, 150 µg/ml) and kept for stirring at 1200 rpm and analyzed by CD spectroscopy after 24 h. 2.3. Preparation, optimization and characterization of insulin loaded LCNPs 2.3.1. Preparation of insulin loaded LCNPs Hydrotope method was used to prepare insulin loaded LCNPs by following the previously reported protocol with slight modifications.16 Briefly, Phytantriol (100 mg) and lecithin (10 mg) were dissolved in ethanol (500 µl) and THF (200 µl) respectively followed by mixing of phytantriol and lecithin solution and named as mixture 1. Insulin (20 mg) was dissolved in 0.01N HCl (1.8 ml) and added drop wise to mixture 1 with continuous stirring at 1200 rpm which resulted in the formation of primary emulsion. The primary emulsion was then added drop wise to Pluronic F-127 solution (0.5% w/v, 10 ml) and kept on stirring at 1200 rpm for 24 h.



2.3.2. Optimization of process variables Concentration of lecithin and surfactant, theoretical drug loading were taken as formulation variables and their effect on particle size, size distribution, zeta potential and entrapment efficiency was evaluated. 2.3.2.1

Lecithin

Lecithin concentration ranging from 5-20% w/w with respect to the total lipid was tested by keeping the insulin loading (5%), surfactant concentration (0.7%) and amount of phytantriol (100 mg) constant. 2.3.2.2 Surfactant Pluronic F-127 was selected based on preliminary compatibility studies and tested in different concentrations (0.3, 0.5, 0.7 and 0.9% (w/v) w.r.t the final volume of dispersion) keeping the insulin loading (5%), amount of phytantriol (100 mg) and lecithin (10%) constant. 2.3.2.3

Drug loading

Theoretical drug loading, ranging from 5-25% w/w, was tested by keeping the amount of phytantriol (100 mg), lecithin (10%) and surfactant (0.5%) constant. 2.3.3. Characterization of insulin loaded LCNPs The insulin loaded LCNPs were characterized for particle size, polydispersity index (PDI), zeta potential and entrapment efficiency.


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2.3.3.1 Particle size, PDI and zeta potential Particle size, PDI and zeta potential were measured by Zeta Sizer (Nano ZS, Malvern Instruments, UK). Particle size and PDI were taken as the average of three (15 measurements at each step) while the zeta potential was taken as the average of 20 measurements. 2.3.3.2 Entrapment efficiency Amount of insulin entrapped into the LCNPs was calculated by direct method by measuring the insulin in definite amount of LCNPs. Gel permeation chromatography was used to separate the free unentrapped insulin from the fraction entrapped within the LCNPs. Briefly, Sephadex G50 (previously hydrated with water for 24 h) was filled in a 30 cm glass column with an inner diameter of 1.5 cm. 200 µl of the formulation was loaded onto the column and eluted with 200 µl fractions of distilled water. The method was validated to precisely separate the fractions containing LCNPs with the fractions containing free insulin. The fractions containing LCNPs were collected and the insulin entrapped within the nanoparticles was extracted using double volume of methanol, analyzed using validated RP-HPLC method and calculated by using the following formula; Entrapment Efficiency=

Amount of Insulin in LCNPs ×100 Amount of Insulin taken initially

2.3.3.3 Shape and morphology of LCNPs Transmission Electron Microscopy (TEM) was used to study the shape and morphology of the LCNPs. Sample preparation for TEM analysis was done by placing a drop of each formulation over the formvar coated grid, dried, stained with phosphotungstic acid (1% w/v) solution and analyzed using TEM (Morgagni 268D. Fei Electron Optics) operated at 120 kV.17



2.4. Freeze drying studies Freeze dried LCNPs were prepared by following our already optimized freeze drying cycle, with slight modifications, comprised of three steps.18 Step I: Freezing, temperature was brought down from 10°C to -60°C for 7 h. Step II: Primary drying, temperature was increased from -60°C to 15°C for 38 h keeping the pressure at 200 Torr. Step III: Secondary drying: the samples were kept at 25°C for 60 min keeping the pressure at 100 Torr. Preliminary screening of different cryoprotectants viz. Lactose, Sucrose, Dextrose and Mannitol was done at fixed (5% w/v) concentration and the effect was evaluated by physical appearance of the cake, redispersibility, particle size upon redispersion, percentage shrinkage and the entrapment efficiency. Shrinkage of the cake was quantified as per earlier report.19 Briefly, crosssectional inner area of the vial (Av) and the cake top surface area (Af) was measured and the degree of shrinkage was calculated by following formula;

100

Based upon preliminary results, mannitol was found optimum and further tested for different concentration ranging from 1-10% w/v and its effect on particle size, PDI and redispersibility was measured. Furthermore, chemical stability of the insulin entrapped within the freeze dried formulation was confirmed by HPLC and native PAGE while the conformational stability was evaluated by CD spectroscopy as per our earlier reports.4, 18 2.5. Stability studies in simulated biological fluids Stability in SGF and SIF is one of the major factors which may affect the in vivo performance of the nanoformulations. Hence stability of the insulin loaded LCNPs were determined in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8) as per our earlier report.18


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Briefly, LCNPs (200 µl) were incubated with 1800 µl of the respective media for 2 h in SGF while 4 h in SIF and the effect on particle size, PDI and entrapment efficiency was observed. 2.6. In vitro release study In vitro release profile was assessed in PBS (pH 7.4). LCNPs equivalent to 1 mg of insulin were suspended in 1 ml of PBS in microcentrifuge tubes and kept at 37°C in a shaker bath (80 rpm). Microcentrifuge tubes in triplicate corresponding to each time points were taken out from the shaker bath and the free insulin was separated from the entrapped insulin and analyzed by RPHPLC method as described in section 2.3.3.2. 2.7. Cell uptake studies Caco-2 cell uptake studies were performed to demonstrate the uptake of LCNPs by the enterocytes. Caco-2 cells were grown and maintained as per our previous protocol.18 Cells were grown in six well plate at a density of 5 × 104 cells/well and incubated with free Insulin-FITC and Insulin-FITC loaded LCNPs at 10 µg/ml for 4 h. Following the incubation, the wells were thoroughly washed with PBS (pH 7.4) to remove the nutrient medium, LCNPs and the free Insulin-FITC attached onto the surface of the cells. Cells were fixed by using glutaraldehyde, which aids in cross linking, and washed thereafter using PBS to remove the excess of glutaraldehyde. The cells were imaged using Confocal Laser Scanning Microscope (CLSM) model Olympus FV 1000. 2.8. In vivo studies 2.8.1. Glucose Estimation Plasma glucose level was determined by glucose oxidase peroxidase method using commercially available kit (Autozyme, Accurex, India) by strictly following the manufacture’s protocol.



2.8.2. Animals Adult female Sprague-Dawley rats (250±30 g) were procured from the central animal facility of the NIPER, India, maintained at standard laboratory conditions with 12 h light/12 dark cycle, and used for planned animal studies. All the experimental protocols were dually approved by Institutional Animal Ethics Committee (Approval no. IAEC/12/101) and performed in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPSCEA), India. 2.8.3. Induction of diabetes Diabetes was induced in animals by intraperitoneal administration of STZ (50 mg/kg) dissolved in ice cold 10 mM citrate buffer pH 4.4 just before the injection. Water containing dextrose 10, 7 and 5% w/v was provided to animals following the first, second and third day of injection to reduce the mortality and housed for 7-10 days. Animals with blood glucose level above 250 mg/dl were considered as diabetic and used for the study. 2.8.4. Pharmacodynamic activity (glucose lowering potential) Diabetic animals were randomly divided into three different groups (n=6). Untreated diabetic animals were taken as a negative control while subcutaneously administered standard insulin solution (5 IU/kg) was taken as positive control. Insulin loaded LCNPs were administered orally by using oral gavage at a dose of 50 IU/kg. Animals were fasted overnight before the intervention and kept on fasting during the study however, had free access to water ad libitum. Blood samples were collected from retro orbital plexus using heparinized capillary tubes under mild anesthesia at various time points (0, 1, 2, 4, 8, 12, 18, 24 h). Plasma was separated immediately after blood collection by centrifugation at 10,000 rpm for 10 min at 4°C and stored


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at -80°C until analysis. The cumulative hypoglycemic effect and pharmacological availability (PA) were determined by using the methods mentioned in previous reports.

20-22

Briefly, blood

glucose levels (% of initial value) versus time profiles were obtained; area above the curve (AAC) and area under the curve (AUC) were determined using trapezoidal method. Cumulative hypoglycemic effect upon treatment was determined by comparing AUC of treatment group with that of untreated diabetic control group.23 Cumulative hypoglycemic effect %

)* +,-./0-/1 1203/-24 45,-.56 )* -./0-7/,- 100 )* +,-./0-/1 1203/-24 45,-.56

Pharmacological availability (PA) of insulin following the oral administration of LCNPs was determined by comparing AAC after oral administration with AAC upon s.c. administration of standard insulin solution with dose correction employing the following equation. PA %

AAC :;10% w/w) resulted in formation of liposomes like spherical


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structures as confirmed by TEM analysis (data not shown). This observation could be attributed to the excess of lecithin making liposome like structures rather than going into the LCNPs. Presence of surfactant not only enhances the miscibility of hydrophilic drug in a hydrophobic environment but also prevents the aggregation of nanoparticles by steric stabilization and helps in preserving the nano size and thus resulting in increased absorption. Thus, from the data it is inferred that 0.5% w/v of Pluronic F-127 is sufficient to cater the purpose of its usage. Practical loading is another important parameter by considering the higher doses required in clinical settings and must be optimized to achieve the sufficient drug load. The practical loading was increased from 5 to 20% of theoretical drug loading however no further increase was observed indicating the saturation of the system at 20% drug loading. Physical instability (aggregation/particle fusion) during storage in suspension form is the major hurdle of nanoparticles which has been overcome by adopting freeze drying. Freeze-drying by itself produces stresses that can destabilize the colloidal suspension of nanoformulations, and may induce aggregation and irreversible fusion of nanoparticles. Hence to provide protection against these stresses, different cryoprotectants were tested and their effect on appearance and different formulation characteristics was tested. Mannitol at 5% w/v was found to be effective in maintaining the critical quality attributes of the formulation and could be attributed to the formation of sufficient glassy matrix that could prevent nanoparticles aggregation and protect them against the mechanical stress. Poor matrix formation, unable to provide sufficient protection in case of other cryoprotectants, could be the best possible explanation of significant changes observed in case of other cryoprotectants. Moreover, the chemical and conformational stability of the insulin entrapped with in the freeze dried formulation was confirmed by HPLC analysis, gel electrophoresis and CD spectroscopy. All the techniques collectively confirmed the



stability of the insulin entrapped with in the freeze dried LCNPs indicative of the suitability of the process parameters to develop the freeze dried LCNPs. Poor stability in the harsh gastric environment is the major hurdle need to be overcome for successful oral delivery. To mimic the harsh gastrointestinal conditions to be faced following oral administration the LCNPs were challenged with simulated biological fluids and the effect was measured in terms of change in size, PDI and percentage of insulin retained within the system. Insignificant change in formulation quality attributes could be attributed to the stability of phytantriol and thus the formed hexagonal LCNPs in simulated biological fluids. Insulin loaded LCNPs demonstrated sustained release for up to 24 h. Slow release observed during initial hours could be ascribed to the time taken by release media to get enter to the hydrophilic channels of LCNPs which latter on dissolved the insulin and showed enhanced release thereafter. The Caco-2 cell is derived from colon carcinoma however, when grown under specific conditions, differentiate and polarize in a manner which resembles the enterocytes lining of the small intestine.

5

To support our hypothesis of enhanced uptake of insulin loaded LCNPs

following oral administration we performed the caco-2 cell uptake study by using Insulin-FITC which revealed significantly higher uptake in comparison with free Insulin-FITC (Figure 7) which was further confirmed by horizontal series line analysis (Figure 7e). In line with the results of higher caco-2 cell uptake LCNPs demonstrated more than double the cumulative hypoglycemia in comparison with subcutaneously administered standard insulin solution. Transient hypoglycemia observed in case of standard insulin solution administered subcutaneously could be attributed to the prompt availability of insulin to systemic circulation while sustained glucose lowering profile observed in case of LCNPs could be attributed to the


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sustained release and thus the availability of insulin entrapped within the LCNPs for therapeutic action. 5.

Conclusion

Overall the developed LCNPs demonstrated excellent stability, remarkably higher uptake and sustained glucose lowering profile. Ease of development and cost effectiveness of the formulation components are the other advantages which make this approach easy to scalable for further studies. Dose response effect and surface functionalization with suitable targeting ligand of this formulation strategy can be further explored. Acknowledgement Authors are thankful to Director, NIPER, for providing infrastructure facilities. AKA and VK are thankful to Council of Scientific & Industrial Research (CSIR), New Delhi, India for fellowship. Technical assistance by Mr. Vinod Kumar in TEM analysis is thankfully acknowledged.



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List of Figures: Figure 1. Preliminary compatibility studies of insulin with (A) Phytantriol (B) solvents and (C) surfactants in terms of conformational stability measured by CD spectroscopy. Figure 2. Surface morphological analysis of freeze dried insulin loaded LCNPs by Transmission Electron Microscopy (TEM). Figure 3. Lyophilized batches of LCNPs with different cryoprotectants at 5% w/v concentration. Figure 4. Lyophilized batches of LCNPs at different concentrations of mannitol (1-10% w/v). Figure 5. Chemical and conformational stability of insulin by comparing the HPLC chromatograms of standard insulin (A) and insulin entrapped with in the freeze dried LCNPs (B) Native PAGE analysis (C) of standard insulin (I) and insulin entrapped with in the freeze dried LCNPs (II) and CD spectroscopic analysis of different insulin samples (D). Figure 6. In vitro release profile of insulin in PBS (pH 7.4). Data have shown as cumulative percentage of insulin released with respect to time. (Data is presented as mean ± SD, n=3) Figure 7. CLSM images showing Caco-2 cell uptake of free Insulin-FITC (A) and Insulin-FITC loaded LCNPs following the 4 h of incubation. Different panels in images represents as; images under the green fluorescence channel (a) differential interface contrast images of Caco-2 cells (b) super impossible image of a and b (c) zoomed image of cells and horizontal line series analysis of fluorescence (e). Figure 8. Plasma glucose level versus time profile of diabetic rats following oral administration of insulin loaded LCNPs (50 I.U./kg) in comparison with std. Insulin given subcutaneously at (5 I.U./kg).


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Page 25 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1: Effect of different concentrations of lecithin on Particle size, PDI, Zeta potential, Entrapment efficiency Lecithin

Particle Size

(% w/w)

(nm)

0

273.91 ± 2.97

5

PDI

Zeta Potential

Entrapment Efficiency

(mV)

(%)

0.232 ± 0.034

-7.25 ± 0.92

16.63 ± 3.16

296.30 ± 2.97

0.234 ± 0.047

-15.85 ± 0.49

42.29 ± 2.21

10

238.25 ± 0.78

0.193 ± 0.003

-20.15 ± 0.07

96.63 ± 0.79

15

238.15 ± 3.75

0.228 ± 0.005

-20.55 ± 0.21

96.78 ± 0.94

20

236.40 ± 3.96

0.231 ± 0.020

-20.75 ± 0.07

98.55 ± 0.67

Values are expressed as mean ± SD (n=6)



Page 26 of 42

Table 2: Effect of different surfactant concentration on particle size, PDI, zeta potential and entrapment efficiency Surfactant

Particle Size

(% w/v)

(nm)

0.3%

276.15 ± 0.35

0.5%

PDI

Zeta Potential

Entrapment Efficiency

(mV)

(%)

0.186 ± 0.007

-19.5 ± 1.84

98.67 ± 1.25

236.73 ± 3.54

0.174 ± 0.032

-20.15 ± 2.19

96.27 ± 1.55

0.7%

238.25 ± 0.78

0.193 ± 0.003

-20.15 ± 0.07

96.63 ± 0.79

0.9%

230.65 ± 2.19

0.093 ± 0.003

-20.5 ± 1.56

92.8 ± 0.10



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Table 3: Effect of different theoretical drug loading on particle size, PDI, zeta potential and practical drug loading Theoretical drug loading (% w/w) 5%

Particle Size (nm)

PDI

236.73 ± 3.54

10% 15% 20% 25%

0.174 ± 0.032

Zeta Potential (mV) -20.15 ± 2.19

Entrapment Efficiency (%) 96.27 ± 1.55

Practical drug loading (%) 4.81 ± 0.08

244.25 ± 0.21 246.15 ± 0.92

0.202 ± 0.008 0.219 ± 0.004

-22.60 ± 0.28 -20.42 ± 0.42

59.74 ± 4.23 48.40 ± 0.53

5.97 ± 0.42 7.26 ± 0.08

245.50 ± 6.36 246.12 ± 3.53

0.220 ± 0.042 0.220 ± 0.009

-18.30 ± 1.27 -14.95 ± 0.21

44.17 ± 1.47 34.39 ± 2.92

8.83 ± 0.29 8.60 ± 0.73




Page 28 of 42

Table 4: Physical appearance of lyophilized batches of LCNPs in the presence of various lyoprotectants Cryoprotectant

Color

Finish

Topography

Structure

Shrinkage

Lactose

White

Matte

Cracks

Dense

12.5%

Sucrose

White

Matte

Cracks

Porous

25%

Dextrose

Cream

Sheen

Collapsed

-

-

Mannitol

White

Matte

Plain

Dense

5.56%


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Table 5: Effect of different lyoprotectants on the particle size, PDI and reconstitution behavior of LCNPs Lyoprotectant

Particle size

PDI

RS

Initial

Final

Ri=Sf/Si

Initial

Final

Lactose

245.45±3.32

273.64±9.89

1.10±0.04

0.234±0.006

0.259±0.01

***

Sucrose

245.45±3.32

267.92±1.56

1.08±0.01

0.234±0.006

0.251±0.006

*

Dextrose

245.45±3.32

-

-

0.234±0.006

-

-

Mannitol

245.45±3.32

249.21±4.80

1.04±0.04

0.234±0.006

0.252±0.012

***

Values are expressed as mean ± SD (n=6) Ri-Redispersibility index, RS-Reconstitution score, (*** redispersible within 20 sec with mere mixing, * redispersion requires high shear vortexing for 2 min, but the cake was not completely redispersed,- completely distorted cake structure was observed hence not measured for size and PDI)



Page 30 of 42

Table 6: Effect of concentration of mannitol on the physical properties of lyophilized batches of LCNPs Mannitol

Color

Finish

Topography

Structure

(% w/v)

Shrinkage (%)

1

White

Matte

Plain

Dense

12.5

2.5

White

Matte

Plain

Dense

11.8

5

White

Matte

Plain

Dense

5.56

7.5

White

Matte

Plain

Dense

5.56

10

White

Matte

Small peaks

Dense

0


Page 31 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 7: Effect of different concentrations of mannitol on the particle size and PDI of lyophilized batches of LCNPs Mannitol (% w/v)

Particle size

PDI

RS

Initial

Final

Ri=Sf/Si

Initial

Final

1

245.4±3.3

250.12±3.68

1.02±0.02

0.234±0.006

0.285±0.009

**

2.5 5 7.5 10

-

250.15±6.29

1.02±0.04

0.234±0.006

0.271±0.013

-

249.20±4.80 243.45±0.92 245.65±0.07

1.01±0.03 0.99±0.01 1.00±0.01

0.234±0.006 0.234±0.006 0.234±0.006

0.252±0.012 0.268±0.019 0.229±0.004

*** *** *** ***

Values are expressed as mean ± SD (n=6) Ri-Redispersibility index, RS-Reconstitution score, (*** redispersible within 20 sec with mere mixing, ** redispersible within 1 min)



Page 32 of 42

Table 8: Stability studies of LCNPs in presence of simulated biological fluids Media

Particle size Initial

SGF

Final

PDI Initial

Entrapment Efficiency (%) Final

Initial

Final

249.5±2.06 251.8±2.84 0.197±0.012 0.210±0.072

44.16±1.47

40.94±0.92

249.5±2.06 245.5±3.63 0.197±0.012 0.239±0.230

44.16±1.47

36.65±0.64

pH 1.2 SIF pH 6.8 Values are expressed as mean ± SD (n=6); Initial indicates the original formulation parameters while the final indicates the formulation parameters following the incubation in SGF and SIF for 2 and 4 h, respectively.


Page 33 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 9: Pharmacodynamic parameters following the administration of standard insulin through subcutaneous route and insulin loaded LCNPs orally. Group

Dose

AUC

AAC

Cumulative

PA (%)

hypoglycemia

(I.U./kg)

Tmax

Cmin

(h)

(% of

(%)

initial value)

Insulin

5

1837.7±34.2

505.9±10.4

21.6±2.1

-

4

13.0±0.9

50

1285.4±25.6 1058.3±17.5

45.1±2.1

20.9±1.5

12

36.5±1.5

s.c. LCNPs

Values are expressed as mean ± SEM (n=6)



Figure 1. Preliminary compatibility studies of insulin with (A) Phytantriol (B) solvents and (C) surfactants in terms of conformational stability measured by CD spectroscopy. 197x46mm (300 x 300 DPI)


Page 34 of 42

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Figure 2. Surface morphological analysis of freeze dried insulin loaded LCNPs by Transmission Electron Microscopy (TEM) 39x33mm (300 x 300 DPI)



Figure 3. Lyophilized batches of LCNPs with different cryoprotectants at 5% w/v concentration 81x41mm (300 x 300 DPI)


Page 36 of 42

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Figure 4. Lyophilized batches of LCNPs at different concentrations of mannitol (1-10% w/v) 81x37mm (300 x 300 DPI)



Figure 5. Chemical and conformational stability of insulin by comparing the HPLC chromatograms of standard insulin (A) and insulin entrapped with in the freeze dried LCNPs (B) Native PAGE analysis (C) of standard insulin (I) and insulin entrapped with in the freeze dried LCNPs (II) and CD spectroscopic analysis of different insulin samples (D) 73x51mm (300 x 300 DPI)


Page 38 of 42

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Figure 6. In vitro release profile of insulin in PBS (pH 7.4). Data have shown as cumulative percentage of insulin released with respect to time. (Data is presented as mean ± SD, n=3) 40x26mm (300 x 300 DPI)



Figure 7. CLSM images showing Caco-2 cell uptake of free Insulin-FITC (A) and Insulin-FITC loaded LCNPs following the 4 h of incubation. Different panels in images represents as; images under the green fluorescence channel (a) differential interface contrast images of Caco-2 cells (b) super impossible image of a and b (c) zoomed image of cells and horizontal line series analysis of fluorescence (e). 49x52mm (300 x 300 DPI)


Page 40 of 42

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Figure 8. Plasma glucose level versus time profile of diabetic rats following oral administration of insulin loaded LCNPs (50 I.U./kg) in comparison with std. Insulin given subcutaneously at (5 I.U./kg). 65x45mm (300 x 300 DPI)


Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


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