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In Vitro and In Vivo Evaluation of Osteoporosis Therapeutic Peptide PTH 1-34 Loaded PEGylated Chitosan Nanoparticles Deepa Narayanan, Anitha A, R. Jayakumar, and K. P. Chennazhi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp400184v • Publication Date (Web): 05 Sep 2013 Downloaded from http://pubs.acs.org on October 1, 2013

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Molecular Pharmaceutics

In Vitro and In Vivo Evaluation of Osteoporosis Therapeutic Peptide PTH 1-34 Loaded PEGylated Chitosan Nanoparticles Deepa Narayanan, Anitha A., R. Jayakumar*, K. P. Chennazhi * Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kochi-682041, India ___________________________________________ *Corresponding Author 1: Dr. R. Jayakumar Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi-682041, Kerala, India Tel: +91-484-2801234; Fax: +91-484-2802020 E-mail: [email protected] & [email protected].

*Corresponding Author 2: Dr. K. P. Chennazhi Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi-682041, Kerala, India Tel: +91-484-2801234; Fax: +91-484-2802020 E-mail: [email protected] & [email protected] 1

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Table of Contents /Graphic abstract

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Abstract Oral formulation of human parathyroid hormone 1-34 (PTH 1-34) is an alternative patient compliant route in treating osteoporosis. PTH 1-34 loaded chitosan nanoparticles was PEGylated (PEG-CS-PTH NPs) and characterised by DLS, SEM, TEM and FTIR. PEGCS-PTH NP aggregates of 200-250 nm which in turn comprised of 20nm individual nanoparticles were observed in SEM and TEM images respectively. The PEG-CS-PTH NP with 40% encapsulation efficiency was subjected to an in vitro release in simulated rat body fluids. PEG-CS-PTH NPs’ treated human primary osteoblast cells upon PTH 1-34 receptor activation, produced second messenger-cAMP which down stream stimulated intracellular calcium uptake, production of bone specific alkaline phosphatase, osteocalcin etc. which substantiates the anabolic effect of the peptide. PEG-CS-PTH NPs showed an oral bioavailability of 100-160pg/mL PTH 1-34 throughout 48 h which is remarkable compared to the bare PTH 1-34 and CS-PTH NPs. NIR image of gastrointestinal transit of ICG conjugated PEG-CS-PTH NPs supports this significant finding. KEYWORDS: PEGylated chitosan nanoparticle, PTH 1-34, improved half life, oral peptide delivery, osteoporosis

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1. INTRODUCTION Osteoporosis is characterized by decreased density, fragility and porosity of bones resulting in frequent fractures.1 Recombinant human parathyroid hormone (PTH 1-34) is approved by FDA for the treatment of osteoporosis.2 This 4117.8 Da peptide induces anabolic responses in bone-forming cells and stimulates bone matrix production and suppresses osteoblast apoptosis.

3, 4

PTH 1-34 (t1/2 1h) is currently administered as a

subcutaneous injection, daily for a two year treatment span.5 Oral route of administration which prolongs the time taken for PTH 1-34 to be exposed to serum can increase half-life of the peptide and improve patient compliance by replacing daily injections. The challenge for such an accomplishment is the ability of the peptide to survive the physiological conditions of the gastrointestinal (GI) tract and deliver therapeutic picogram levels of peptide in the systemic circulation. PTH 1-34 is entirely degraded by trypsin, chymotrypsin and pepsin within 5 minutes and rat small intestinal mucosa degrades approximately half of the peptide in 3 h .6 This precise characterisation of the enzymatic barrier for oral PTH 1-34 administration justifies the need for development of PTH 1-34 delivery systems. The use of nanosystems as delivery agents confers advantages such as simple and cost effective synthesis procedures, use of minimal peptide concentrations for encapsulation, protection of the peptide in the GI tract, efficient absorption of the nanoformulation across the microvillus, prolonged circulation and slow pH dependent release of anabolic dose of peptide .7-12 Chitosan (CS), a biopolymer finds immense applications in biomedical fields because of its biocompatibility and non toxicity.

13

Co polymerisation of glucosamine and

N-acetylglucosamine yields this naturally occurring cationic aminopolysaccharide. It 4


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originates from shells of crustaceans and is the second most abundant natural polysaccharide. CS is non-toxic mucoadhesive biopolymer that is biocompatible. Certain biocompatible polyanionic substances such as sulfate, citrate, and tripolyphosphate can act as cross linkers in the preparation of biodegradable nano/microparticles of CS. CS has been proved to promote ordered regeneration of soft tissue and osteoinduction, for therapeutical correction of osteoporosis in the elderly.14-16 Nanoparticles of CS (CS NPs) exhibits strong electrostatic interaction with peptides and proteins, protecting them from enzymatic degradation and permitting its paracellular uptake making it suitable for oral delivery of PTH 1-34.17-24 We have earlier reported the synthesis of 60-80 nm sized PTH 1-34 loaded CS NPs.24 These CS-PTH NPs due to the exposed amine groups may favor higher dissolution in the gastric pH and could also result in

hemolysis if the CS

concentration is increased while administering a dose . Due to which using this system for in vivo oral delivery is limited. To overcome this drawback, modifying the surface with polyethylene glycol (PEG) was the appropriate solution since PEGylation of nanoparticle is reported to increase stability in gastric and biological fluids which can prolong the particles circulation and retard elimination. PEGylation can also facilitate the transport of bioactive

macromolecules

across

the

intestinal

epithelium

and

improve

drug

bioavailability in vivo.25-28 The absorption-molecular weight profiles examined using different-sized PEGs were different between the small and large intestines; the largeintestinal absorption of PEGs with molecular weights larger than 300 was poor, while PEGs with molecular weights up to 600 were relatively well absorbed in the small intestine.29 So to ensure maximum absorption low molecular weight PEG 200 is ideal.

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Hence in the present study we used PEG 200 to synthesise PEG-CS-PTH NPs and after characterisation performed in vitro biological assays and drug release, prior to investigating its potential as an oral peptide delivery vehicle in Sprague dawley rats.

2. EXPERIMENTAL SECTION 2.1. Materials Chitosan (Mol Wt 100-150 KDa and degree of deacetylation-80%) was purchased from Koyo chemical Co Ltd; Japan. PTH 1-34 pure lyophilised 0.5 mg pellets (Mol Wt 4117.8 Da) was purchased from Genscript USA. Poly (ethylene glycol) (PEG-200 Mw), Penta sodium tripolyphosphate (TPP), MTT (3-(4, 5-Dimethylthiazole-2-yl) - 2, 5 diphenyl tetrazolium) and Bicinchoninic acid assay (BCA) reagents was purchased from Sigma Aldrich. Lactate dehydrogenase assay (LDH assay) kit and PTH 1-34 ELISA kit was purchased from ALPCO diagnostics. Bone specific alkaline phosphatase assay (bAP Assay) kit was purchased from Krishgen Biosystems, osteocalcin ELISA from Immunodiagnostic systems USA, calcium calorimetric assay was purchased from Bio Vision and cAMP assay was purchased from Assay designs USA. Primary human osteoblast and the osteoblast specific media were purchased from Promo cell. 2.2. Preparation of PEG-CS-PTH NPs 10 mL CS solution of 0.1 % (w/v) was prepared in 0.3 % acetic acid into which 0.5 mg PTH 1-34 (5x10-2 mg/mL or 10-5 M) was added and incubated at 4º C for 4 h under constant stirring. To this mixture 1.0 % TPP was added drop wise to obtain a nanosuspension. PEG 200 was added to the polymer in a 1:2 w/w ratio and stirred for

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another hour. The suspension obtained thereafter was centrifuged at 15,000 - x g for 20 minutes. The pelleted particles were resuspended in deionised water. 2.3. Characterization The mean size and zeta potential of the PEG-CS-PTH NPs were determined by photon correlation spectroscopy using a Zeta Plus particle analyzer (Brookhaven Instrument Corp., Holtsville, NY, USA).The surface morphology was evaluated using Scanning electron microscope (SEM) ; JEOLJSM-6490LA. Size and shape of the nanoparticles were further characterised using the transmission electron microscope (TEM); JEOL JEM 2100. Fourier Transform Infrared Spectroscopy (FT-IR) spectrum was used to identify the potential interaction between PEG, CS and PTH 1-34 in the nanoparticles

using

Perkin

Elmer

Spectrum

RXI

Fourier

Transform

Infrared

spectrophotometer using KBr method. 2.4. Entrapment efficiency and loading efficiency The amount of PTH 1-34 entrapped into the nanoparticles was quantified using ELISA kit and the percentage loading was calculated according to the previous reports.30 2.5. In vitro peptide release studies In vitro PTH 1-34 release from PEG-CS-PTH NPs was determined at pH 7.5, 6.8 and 3.4. The nanoparticles suspension was pelletized and redispersed in 10 mL of 0.9% saline with a final peptide concentration of 20µg/mL. From this 1mL nanoformulation was added to 20 mL of PBS at pH 7.5 corresponding to the pH and volume of rat blood. Another 1mL was added to 3.4mL simulated rat gastric fluid pH 3.4 and simulated rat intestinal fluid pH 6.8 (enzyme free) corresponding to that of overnight starved rats.31 Beakers were incubated at 37 °C under gentle shaking. At definite time intervals, 1mL of 7



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the release solution was collected and replaced with the fresh solutions. The sample was centrifuged and the supernatant was analysed using PTH 1-34 ELISA kit. Amount of PTH 1-34 released at time “t” XEq. 100(1)

Peptide release % = Amount of PTH 1-34 loaded in the nanoformulation

Thereafter a cumulative graph of the percentage of peptide released was plotted against each time period. 2.6. APTT/PT The coagulating effect of drug loaded nanoparticles and controls on plasma was determined by plasma coagulation assay, which is performed by two tests, i.e., prothrombin time (PT) and activated partial thromboplastin time (APTT). Previously published protocol was followed for the experiment.32 2.7. Haemocompatibility studies To 1mL of blood sample, 100 µL of PEG-CS-PTH NPs with PTH 1-34 concentrations of 20 and 10 µg/mL was added. Triton –X 100 (1%) and saline were the positive and negative controls respectively. Each concentration was evaluated in triplicate. Thereafter the blood compatibility was evaluated as previously reported.24 2. 8. Biocompatibility studies Primary human osteoblast cells were maintained in osteoblast growth media. The cells were incubated in 5 % CO2 incubator at 37 °C. After attaining confluency, the cells were detached from the flask. The cell suspension was centrifuged at 3000 rpm for 3 minutes and then resuspended in media for further studies. To test the biocompatibility of the synthesised nanoparticles (MTT and LDH assay), primary human osteoblast cells 8


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was seeded at a density of 10,000 cells/well into a 96 well plate. After attaining 90% confluency, the cells were incubated with (a) 0.05 mg/mL, (b) 0.005 mg/mL, (c) 0.0005mg/mL PTH 1-34 loaded PEGylated CS NPs and chitosan nanoparticle concentrations corresponding to the PTH 1-34 loaded concentrations, (d) 0.1 mg/mL, (e) 0.01 mg/mL, (f) 0.001 mg/mL along with (g) media and (h) triton –X 100 for a period of 24 h. Triplicate samples were analysed for each experiment as per the previously published protocol for the MTT assay.24 LDH analyses were done in accordance to the manual provided with the assay kit. Toxicity of the samples was expressed as the percentage of the positive control (1 % Triton X-100) calculated as % Toxicity = (Nt/Nc) ×100

Eq. (2)

Nt is the absorbance of cells treated with sample and Nc is the absorbance of the cells treated with 1 % Triton-X 100. 2.9. Biological assays 50,000 cells per well of 6 well plates was seeded with primary human osteoblast and on attaining 70% confluence were treated in triplicates with PEG-CS-PTH NPs, CS NPs and PTH 1-34 for a period of 7 days. Cells untreated were kept as controls. Thereafter the following biological assays were performed. 2.9.1. Estimation of total protein by BCA assay and in vitro bone specific alkaline phosphatase activity (bAP) The estimation of total protein and bAP abides by the protocol reported previously.24

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2.9.2. Calcium calorimetric assay Ca2+ sequestration and release into and out of the cytoplasm, functions as a signal for many cellular processes. Depending on the level of PTH 1-34 in the extracellular medium, Ca2+ may be effluxed or influxed by the primary human osteoblast.The cell lysate was analyzed using the calcium calorimetric assay kit for quantifying the intracellular calcium. 2.9.3. Osteocalcin assay The detection of extracellular levels of osteocalcin produced and released by primary human osteoblast is valuable for the study of bone metabolism. The media used for the experiments is collected and analysed for osteocalcin using the specific ELISA kit and quantified using the instruction manual. 2.9.4. cAMP assay The cells were lysed with 0.1M HCl, centrifuged and the supernatant was lyophilised. Samples were assayed following the instructions in the manual provided along with the kit and the cAMP concentrations are determined. 2.9.5. Cell proliferation Light microscope images of primary human osteoblast cells after exposure to PEG-CS-PTH NPs for 7 days was observed, photographed and compared with controls. 2.10. In vivo experiments The use of female Sprague dawley for the in vivo experiments was approved by the institutional ethical committee for animal studies.All the experiments were carried out following the approved protocols for the pharmacokinetic evaluation of oral uptake of 10


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PTH 1-34 from PEG-CS-PTH NPs. The PEG-CS-PTH NPs was administered through oral gavage in one set of animals; n=3 (animals were subjected to an overnight fast). Blood was drawn (~150 µl) by cannulation at different time intervals from retro orbital sinus. The serum separated was immediately analysed using Human PTH 1-34 ELISA kit. The results were compared with control PTH 1-34 and CS-PTH 1-34 NPs administered orally. The cross reactivity of endogeneous rat PTH 1-34 was also measured. In vivo imaging using near infra red imaging system of rats orally administered with PEG-CS-PTH NPs conjugated to indocyanin green helped to track the path and residence time of formulation in the gastrointestinal tract. 2.11. Statistical analysis All the above experiments were conducted three times independently with triplicates and the results were expressed as mean ± standard deviation. The standard deviation values are indicated as error bars in the corresponding graphs. Student’s Ttest was performed to find the statistical significance of the values. A probability of p< 0.05 was considered to be statistically significant.

3. RESULTS 3.1. Preparation and Characterization of PEG-CS-PTH NPs SEM image of PEG-CS-PTH 1-34 NPs(200-250nm) is shown in Fig.1 A. TEM image proves that such an aggregate (Fig.1 B) is in turn formed of individual nanoparticle of approximately 20 nm in diameter (Fig.1 C and D).33 Zeta potential measurement of +35 indicates the stability and surface charge of the PEG-CS-PTH NPs.

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Figure1. (A) SEM of PEG-CS-PTH NPs showing particle size of 200-250 nm(B) TEM image of a 200-250nm PEG-CS-PTH NPs aggregate

(C) and (D) TEM image

showing that the cluster of PEG-CS-PTH 1-34 NPs are in turn formed of individual particles of approximately 20nm. Fig. 2 shows the FT-IR spectra of PEG, CS-PTH NPs and PEG-CS-PTH NPs. The PTH 1-34 loading on chitosan nanoparticles was confirmed by a sharp peak at 980 and 3450 cm-1, which is indicative of the presence of the excess –NH2 and –COOH groups contributed by the amino acids from PTH 1-34. Moreover the existence of 980 cm-1 peak in the observed FT-IR spectrum can be considered as a peak for the chitosan content in the peptide loaded nanoformulation. The 2857 and 2924 cm-1 peak is intense 12


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and it represents abundant -CH2 residues of the loaded PTH 1-34 moieties.24 The stretching frequencies for major functionalities have been shifted to lower frequency regions, as the TPP has interacted effectively with the protonated amine or amide residues in the nanoformulation. The stretching frequency is directly proportional to the wave number. PEG coats the positively charged nanoparticle by electrostatic interaction. The –NH3+ bending at 1570 cm-1observed in the CS-PTH NPs spectra has narrowed which proves the interaction of -NH3+ with the -OH groups of PEG. 2850-2960 cm-1 in the spectra of PEG marks the presence of alkanes in PEG, a similar less intense peak is observed at the same position in PEG -CS-PTH NPs but not in CS-PTH NPs spectra. This shows the contribution of alkanes by PEG in the PEG-CS-PTH NPs.33, 34

Figure 2. FTIR of the CS-PTH NPs, PEG-CS-PTH NPs and PEG 200. 3.2. Entrapment efficiency, loading efficiency and in vitro peptide release studies The entrapment efficiency of PEG-CS-PTH NPs with 0.1 w/v CS and 0.05mg/mL PTH 1-34 was 40 % and the yield of nanoparticles 14.2 mg /15mg based on which the 13



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loading efficiency was calculated to be 1.408 %. Fig. 3, shows the cumulative in vitro release profile of PTH 1-34 at pH 3.4, pH 6.8 (volume 3.4ml), and pH 7.5 (volume 20ml) from PEG-CS-PTH NPs. The cumulated release at the end of 2h at pH 3.4 was 2500pg/mL (Fig. 3, A). Fig. 3, B shows a cumulated release of 3000pg/mL only at the end of 24h at pH 6.8. Fig. 3, C shows the cumulative in vitro release of 5000-6000 pg/mL of PTH 1-34 at pH 7.5 (volume 20ml) from PEG-CS-PTH NPs which was carried out for a period of 48h.

Figure 3. Cumulative in vitro release of PTH 1-34 from the PEG-CS-PTH NPs at pH 3.4, pH 6.8, and pH 7.5 and which is the gastric, intestinal and blood pH of rats subjected to overnight fast. 14


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3.3. Hemocompatibility studies and biocompatibility studies The APTT and PT results (Fig. 4, A, B) showed that the PEG-CS-PTH NPs have not affected the intrinsic and extrinsic clotting pathways. Hemocompatibility is a significant index for biomaterials since these materials when exposed to blood may damage the erythrocytes in certain degree or cause the formation of thrombus. Haemolysis assay was carried out to evaluate the blood compatibility of relevant concentrations PEG-CS-PTH NPs .Damage of the erythrocytes due to the sample was insignificant as plasma haemoglobin content detected in the sample treated blood was way below the detection limits of the spectrophotometer (BioTek Power Wave XS) and that of the positive control was 88 % (data not shown ).

Figure 4. Blood compatibility results of the PEG-CS-PTH NPs and CS NPs (A) APTT and (B) PT 15



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The MTT and LDH assays (Fig. 5, A, B) proved the biocompatibility of samples on primary human osteoblast

cells.

Figure 5. Biocompatibility assays (A) MTT assay; (a) 0.05 mg/mL, (b) 0.005 mg/mL, (c) 0.0005mg/mL PTH 1-34 loaded PEGylated CS NPs and chitosan nanoparticle concentrations corresponding to the PTH 1-34 loaded concentrations, (d) 0.1 mg/mL, (e) 0.01 mg/mL, (f) 0.001 mg/mL along with (g) media and (h) triton –X 100 (B) LDH assay (a) 0.05 mg/mL, (b) 0.005 mg/mL, (c) 0.0005mg/mL PTH 1-34 loaded

PEGylated

CS

NPs

and

chitosan

nanoparticle

concentrations

corresponding to the PTH 1-34 loaded concentrations, (d) 0.1 mg/mL, (e) 0.01 mg/mL, (f) 0.001 mg/mL along with (g) media and (h) triton –X 100. 16


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3.4. Total protein estimation (BCA assay) and in vitro alkaline phosphatase activity Fig. 6, A compares the 7 day BCA assay results of primary human osteoblast cells treated with PEG-CS-PTH NPs, CS NPs, PTH 1-34 and cells alone. 20µg/mL PTH 1-34

concentrations in the PEG-CS-PTH NPs have released sufficient peptide to

stimulate production of 0.3mg/mL proteins in 7days which is 0.4mg/mL in the case of PTH 1-34 treated cells and there is a dip to 0.2mg/mL in the case of cells alone and that treated with CS NPs. Peptide loaded nanoparticles have released sufficient PTH 1-34 to stimulate the production of 1600ng/mL bAP in the 7-day experiment span (Fig.6 B). Concentration of bAP produced by the primary human osteoblast

treated with the

peptide alone was 2400ng/mL which shows the peptides ability to stimulate bAP production in the primary human osteoblast cells. Cells grown as controls and those treated with CS NPs have released 1200ng/mL bAP which is the basic quantity this population of primary human osteoblast

cells can produce in 7 days. The test and

controls of experiment conducted for 3 day span have more or less the same quantity of bAP produced which is 400ng/mL.

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Figure 6. (A) BCA assay showing the total protein produced by primary human osteoblast cells after 3 days and 7 days (B) 3 and 7 day bone specific alkaline phosphatase detected by the bAP assay. # indicates the p< 0.05 when compared to 3 day PEG-CS-PTH NPs treated primary human osteoblast cells and *indicates p value< 0.05 when compared to 7 day PEG-CS-PTH NPs treated primary human osteoblast cells. 3.5. Calcium calorimetric assay, Osteocalcin assay and cAMP assay The calcium calorimetric assay (Fig. 7, A) shows the influx of calcium due to the effect of PTH 1-34. The cells treated with PEG-CS-PTH NPs had 28.09 µg/mL intracellular calcium whereas the rest of the treated control cells had less than 15 µg/mL intracellular calcium. The obtained result along with the cell proliferation data indicates possible anabolism in osteoblast cells. The level of osteocalcin produced by PEG-CS18


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PTH NPs treated primary human osteoblast cells (Fig. 7, B) is nearly 3 ng/mL which is only a slight increase compared to the other control groups (between 2 and 2.5 ng/mL). 1.5 -2 pgmol/mL cAMP, a second messenger is produced in response to PTH 134 as shown in Fig.7, C .This heightened level of cAMP in peptide and peptide loaded nanoparticle treated primary human osteoblast cells compared to ≤ 0.5pgmol/mL in the CS NPs and cells in media alone indicates the hormonal stimulation of the transmembrane PTH 1-34 receptors on the primary human osteoblast and the extend of signalling happening within the cells. This pathway is the most crucial in deciding the anabolic effect of PTH 1-34 on primary human osteoblast cells.

Figure 7. (A) Calcium assay, (B) Osteocalcin assay and (C) cAMP assay. *indicates p value< 0.05 when compared to 7 day PEG-CS-PTH NPs treated primary human osteoblast cells 19



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3.6. Cell proliferation The cell number in the wells treated with PEG-CS-PTH NPs were visibly high and was arranged in concentric pattern (Fig. 8) which could be the influence of the peptide .The data was compared to cells grown in media and those treated with bare PTH 1-34 and CS NPs.

Figure 8. Light microscope image primary osteoblast cells after 7 days (A) grown in osteoblast specific media (B) after treating with PTH 1-34 shows increased cell number (C) after treating with CS NPs(D1-D4) after treating with PEG-CS-PTH NPs shows high cell number and osteoblast specific concentric arrangement of the cells is pointed by the arrows. 20


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3.7. In vivo experiments The Human PTH 1-34 ELISA results of the blood samples collected from the Sprague dawley rats at various time points after the oral administration (Fig. 9) shows that the PEG-CS-PTH NPs efficiently delivers and maintains 100-120pg/mL PTH 1-34 throughout the 48h period of study. CS-PTH NPs is observed to be efficient in delivering 100-160pg/mL PTH 1-34 from the 5th minute to 30 minutes and thereafter the level falls to negligible levels by 1h. There was an observed hemolysis in the initial half an hour of the blood samples collected after oral administration of CS-PTH NPs. Bare PTH 1-34 (20µg/mL) administered orally and the endogenous PTH 1-34 in naive rat that has a cross reactivity with the human PTH 1-34 ELISA kit shows a concentration less than 30pg/mL throughout the study period which proves that there is no intestinal uptake of bare PTH 1-34 from the digestive tract of rat. The point of interest is the detection of PTH 1-34 in the rat blood within 5 minutes of oral administration of the nanoformulation and a maintained steady serum concentration for 48 h in the case of PEG-CS- PTH NPs (Fig. 9 ).

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Figure 9. The pharmacokinetic profile of PTH 1-34 after oral administration of the PEG-CS-PTH NPs, CS-PTH NPs, PTH 1-34 (overnight fast) (*indicates p value< 0.05

in Female Sprague dawley rats when compared to bare PTH 1-34

administered orally at all the different time points). The NIR image of the rats (Fig. 10), orally administered with (ICG) indocyanin green conjugated PEG-CS-PTH NPs revealed its entry into the absorptive site, the intestine within 30 minutes. The mucoadhesive property of the nanoformulation is quite evident as the IR emission of the ICG-PEG-CS-PTH NPs is observed even after 24 hours in the gastrointestinal tract.

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Figure 10. In vivo NIR image of rats orally administered with ICG-PEG-CS-PTH NPs. The areas marked A & B are the junction of stomach and duodenum and the small intestine respectively.

4. DISCUSSION PEG-CS-PTH NPs aggregates of 200-250nm which in turn is formed of 20nm individual particles successfully entrapped and released bioactive

PTH 1-34.

Biocompatibility and hemocompatibility of the formulation was proved. The percentage haemolysis of the sample was far below 5 %, the critical safe haemolytic percentage for biomaterials according to ISO/TR 7406. PEG-CS-PTH NPs (loaded with 20 µg/mL PTH 1-34) released peptide in anabolic dose which was evident from the cell proliferation and concentric patterns 23



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observed in the primary human osteoblast cells. The levels of bAP, total protein, osteocalcin and cAMP produced following a 7 day treatment with the peptide loaded nanoformulation justified the same. An increase in total protein concentration could be due to the production of bone specific proteins like osteocalcin, or due to cell proliferation as an effect of exposure to PTH 1-34. Higher level of intracellular calcium detected in primary human osteoblast treated with the PEG-CS-PTH NPs marks the beginning of bone mineralization. Shi et al have proved that chitosan-coated iron oxide nanoparticles have an effect on osteoblast proliferation and differentiation when compared to the bare iron oxide nanoparticles. Enhanced osteoblast viability, higher alkaline phosphatase activity and extracellular calcium deposition was observed in the cells treated with chitosan coated iron oxide nanoparticles compared with the controls.35 The in vitro release profile of the formulation at pH 3.4, 6.8, and 7.5 indicates its stability and cumulative release pattern. The mechanism of in vitro release is assumed to be the imbibition of water and swelling of the PEG-CS-PTH NPs making the cross linker interaction feeble, resulting in the release of peptide. The PTH 1-34 release at pH 3.4 (Fig. 3,A) was conducted only up to 2h as gastric clearance of empty stomach falls between a minimum of 30minutes to a maximum of 2h. The release at pH 6.8 was extended to 24h as the tendency of intestine is to retain substances this long. But when compared to the 2h release at pH 3.4, less than 1000pg/mL was only released at pH 6.8 at this time point. Thus, showing that the acidic pH has a greater influence on the rate of release of the peptide from the nanoformulation due to its dissolution. But the concentration of peptide loss that occurs in these different pH conditions is negligible when the entrapped amount of 20µg is taken into consideration. 1mL of CS-PTH NPs 24


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orally administered in rats had a CS concentration of 1mg/mL which was observed to initiate haemolysis for the first half an hour following which the condition cleared. The high positive charge on the CS-PTH NPs interacts with the negatively charged sialic acid residues of RBC and causes the clumping and haemolysis. The animals were kept under observation and no further ill effects were observed.PEG-CS-PTH NPs showed no such hemolytic effects after oral administration. PTH 1-34 was detected in the blood of female Sprague dawley rats, following oral administration of PEG-CS-PTH NPs, throughout the 48 h period of study. The NIR images support the fact that the nanoformulation has exhibited high mucoadhesion even beyond 24h. The speedy gastric emptying into the intestine is also evident in the images that prevent the deterioration of peptide loaded nanoformulation in the stomach. Once the formulation is adhered to mucus that lines the gastrointestinal tract, the significant presence of PTH 1-34 in blood could be attributed to the paracellular uptake of the peptide loaded nanoformulation as such or the in situ released peptide (Fig. 11, A). The justification for the peptide uptake is that the water molecules form a close, hydrogen-bonded layer with the soluble PTH 1-34 backbone as well as its hydrophilic side chain amino acid groups. Transcellular PTH 1-34 permeation would involve breakdown of these hydrogen bonds .Overcoming the energy requirement for this process might be an important determinant of transcellular peptide diffusion which would involve the desolvation of the PTH 1-34, whereas the alternate paracellular mode doesn’t require the solute to leave the aqueous environment and, hence depends predominantly on size-sieving of the molecules.36

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Figure 11. (A) Schematic representation of intestinal uptake of the PEG-CS-PTH NPs (B) graphical representation of the supply of the PTH 1-34 released from the nanoformulation through the blood to osteoporotic site showing osteoblast cells surviving and dividing under the influence of anabolic dose of PTH 1-34 (C) diagram depicting the molecular mechanism of the anabolic effect of PTH 1-34 on osteoblast cell. Based on perfusion studies with hydrophilic solutes inulin, glucose, and creatinine; paracellular pores are estimated to have a pore radius of 50 A with a cross sectional area per unit path length of 4.3 cm per cm length of intestine.37 Another interesting observation is the higher permeation of hydrophilic peptides with net positive, rather than 26


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negative charge.38 Partition coefficient (P) of the drug molecule between n-octanol and water is also an important factor determining the mode of peptide and protein absorption through the intestine. Log P values less than zero indicates a hydrophilic molecule, which is more likely to follow the paracellular pathway which uses aqueous channels for molecular diffusion of -3.10

40

39

The 4117 Da low molecular weight, linear PTH 1-34 with a P value

and a net positive charge has a high probability to follow the paracellular

pathway of absorption from the intestinal lumen hence making its presence in rat blood merely after 5 minutes of oral administration. The prolonged circulation of PTH 1-34 might be due to the nanoformulation being absorption into the lacteals in the microvillus. The higher porosity of lymphatic capillaries compared to blood vessel endothelium directs even macromolecules to lymphatic circulation. The interest in lymphatic absorption is linked to the fact that it outflanks the hepatic first-pass metabolism that any blood-absorbed component must undergo, in this way elevating bioavailability.41 The nanoformulation is expected to have survived the gastric environment of the overnight fasted rat stomach (fasting pH of 3.4) due to PEGylation of the nanoformulation as well as the speedier gastric emptying. PEG also has the property to enhance paracellular transport.42 The surface charge of PEG-CS-PTH NP is +35 hence will bind to cell membranes .The positive charges on the CS, interacts with tight junction protein followed by redistribution of filamentous actin, and superficial destabilization of the plasma membrane43,

44

and decrease the trans-epithelial electrical resistance of cell

monolayers to increase paracellular permeability.45,

46

The proposed mechanism of

action of the released PTH 1-34 (Fig. 11, B, and C) is compiled and concluded from earlier work by researchers in the field. The diagram shows the seven transmembrane 27



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receptor stimulated by PTH 1-34 in anabolic dose released from an orally administered PEG-CS PTH 1-34 and a series of events triggered thereafter.3 CS is also reported to have properties of osteoblast adherence and stimulates its proliferation.47, 48 This work proves that PEG-CS-PTH NPs has successfully protected and delivered 100-120pg/mL PTH 1-34 orally in rats. These findings affirmatively imply the potential of this system to release biologically functional levels of peptide in the in vivo rat models used here.49-51 The anabolic action of such a sustained low dose of PTH 1-34 in osteoporotic rat models or other relevant higher animal models will be insightful to conclude the therapeutic efficiency of this orally administered PEG-CS-PTH nanoformulation.52, 53 Notes The authors declare no competing financial interest. Acknowledgement The authors are thankful to Department of Biotechnology (DBT), Govt. of India, for their financial support for this work under the Nanoscience and Nanotechnology Initiative program (Ref. No. BT/PR10882/NNT/28/142/2008).We are thankful to Council of Scientific and Industrial Research (CSIR), India for providing Senior Research Fellowship (SRF award-Ref.No. 9/963(0024)2K12-EMR-I) to the author for carrying out this research work. The authors are also thankful to Koyo Chemicals Co. Ltd., Japan for providing Chitosan.

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List of Figure Legends Fig.1. (A) SEM of PEG-CS-PTH NPs showing

particle size of 200-250 nm(B) TEM

image of a 200-250nm PEG-CS-PTH NPs aggregate (C) and (D) TEM image showing that the cluster of PEG-CS- PTH 1-34 NPs are in turn formed of individual particles of approximately 20nm. Fig.2. FTIR of the CS-PTH NPs, PEG-CS-PTH NPs and PEG 200. Fig.3.

Cumulative in vitro release of PTH 1-34 from the PEG-CS-PTH NPs at pH 3.4, pH 6.8, and pH 7.5 and which is the gastric, intestinal and blood pH of rats subjected to overnight fast.

Fig.4. Blood compatibility results of the PEG-CS-PTH NPs and CS NPs (A) APTT and (B) PT. Fig.5. Biocompatibility assays (A) MTT assay; (a) 0.05 mg/mL, (b) 0.005 mg/mL, (c) 0.0005mg/mL PTH 1-34 loaded PEGylated CS NPs and chitosan nanoparticle concentrations corresponding to the PTH 1-34 loaded concentrations, (d) 0.1 mg/mL, (e) 0.01 mg/mL, (f) 0.001 mg/mL along with (g) media and (h) triton – X 100 (B) LDH assay (a) 0.05 mg/mL, (b) 0.005 mg/mL, (c) 0.0005mg/mL PTH 1-34 loaded PEGylated CS NPs and chitosan nanoparticle concentrations corresponding to the PTH 1-34 loaded concentrations, (d) 0.1 mg/mL, (e) 0.01 mg/mL, (f) 0.001 mg/mL along with (g) media and (h) triton –X 100. Fig.6. (A) BCA assay showing the total protein produced by primary human osteoblast cells after 3 days and 7 days (B) 3 and 7 day bone specific alkaline phosphatase detected by the bAP assay. # indicates the p< 0.05 when compared to 3 day PEG-CS-PTH NPs treated primary human osteoblast cells and *indicates p 37



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Page 38 of 39

value< 0.05 when compared to 7 day PEG-CS-PTH NPs treated primary human osteoblast cells. Fig.7. (A) Calcium assay, (B) Osteocalcin assay and (C) cAMP assay. *indicates p value< 0.05 when compared to 7 day PEG-CS-PTH NPs treated primary human osteoblast cells. Fig.8. Light microscope image primary osteoblast cells after 7 days (A) grown in osteoblast specific media (B) after treating with PTH 1-34 shows increased cell number (C) after treating with CS NPs (D1-D4) after treating with PEG-CS-PTH NPs shows high cell number and osteoblast specific concentric arrangement of the cells is pointed by the arrows. Fig.9. The pharmacokinetic profile of PTH 1-34 after oral administration of the PEG-CSPTH NPs, CS-PTH NPs, PTH 1-34 in Female Sprague dawley rats (overnight fast) (*indicates p value< 0.05 when compared to bare PTH 1-34 administered orally at all the different time points ). Fig.10. In vivo NIR image of rats orally administered with ICG-PEG-CS-PTH NPs .The areas marked A and B are the junction of stomach and duodenum and the small intestine respectively. Fig.11. (A) Schematic representation of intestinal uptake of the PEG-CS-PTH NPs (B) graphical representation of the supply of the PTH 1-34 released from the nanoformulation through the blood to osteoporotic site showing osteoblast cells surviving and dividing under the influence of anabolic dose of PTH 1-34 (C) diagram depicting the molecular mechanism of the anabolic effect of PTH 1-34 on osteoblast cell. 38


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Graphic for manuscript 254x190mm (96 x 96 DPI)


In Vitro and in Vivo Evaluation of Osteoporosis ... - ACS Publications

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