Formulation and evaluation of tacrolimus-loaded galactosylated Poly(lactic-co-glycolic acid) nanoparticles for liver targeting.

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Research Paper

Journal of Pharmacy And Pharmacology

Formulation and evaluation of tacrolimus-loaded galactosylated Poly(lactic-co-glycolic acid) nanoparticles for liver targeting Nishita P. Mistry, Jagruti L. Desai and Hetal P. Thakkar Pharmacy Department, Faculty of Technology and Engineering, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India

Keywords asialoglycoprotein receptors; conjugation; galactose; liver targeting; poly (lactide-co-glycolic acid) Correspondence Nishita P. Mistry, Pharmacy Department, Faculty of Technology and Engineering, The Maharaja Sayajirao University of Baroda, 390 001 Vadodara, Gujarat, India. E-mail: [email protected] Received January 21, 2015 Accepted March 29, 2015 doi: 10.1111/jphp.12430

Abstract Objective The aim of this investigation was to formulate liver targeted tacrolimus-loaded nanoparticles for reducing renal distribution and thereby decreasing nephrotoxicity. Method Poly lactic-co-glycolic acid (PLGA) was galactosylated, and confirmation of galactosylation was performed by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. Tacrolimus-loaded PLGA nanoparticles (Tac-PLGA NP) and galactosylated PLGA nanoparticles (Tac-Gal-PLGA NPs) were prepared by ultrasonic emulsification solvent evaporation technique and characterized. Key findings The size of both the formulations was below 150 nm and negative zeta potential indicated the stability and reticuloendothelial system targeting efficiency. The in-vitro release and pharmacokinetics showed sustained release of tacrolimus from nanoparticles in comparison to plain drug solution. The biodistribution studies revealed the potential of both the nanoparticulate systems to target tacrolimus to the liver for prolonged periods of time compared with the plain drug solution. However, significantly higher liver and spleen targeting efficiency of Tac-Gal-PLGA NPs compared with Tac-PLGA NPs was evident indicating its active targeting. Significantly lower distribution in the kidney from nanoparticles indicated the possibility of reduced nephrotoxicity – the principal reason for patient non-compliance. Both nanoparticles showed stability at refrigerated condition (5°C ± 3°C) upon storage for 1 month. Conclusion Galactosylated PLGA nanoparticles seem to be a promising carrier for liver targeting of tacrolimus.

Introduction Organ transplantation is a surgical procedure to replace a failing or a diseased organ with a healthier donor one. Tissue typing (a group of procedures in which the tissues of a prospective donor and recipient are tested for compatibility before organ transplantation) ensures that the organ or tissue to be transplanted is as similar as possible to the tissues of the recipient. However, the match is usually not perfect since no two people (except identical twins) have identical tissue antigens. The body’s immune system is able to recognize the difference between its own cells and foreign matter. This can trigger transplant rejection, a process in which the recipient’s immune system attacks the transplanted organ or tissue. People with suppressed immune system are less likely to reject their transplanted organs.

Various drugs are used to suppress the immune system to prevent it from attacking the newly transplanted organ.[1] Various classes of drugs such as calcineurin inhibitors, antiproliferative drugs, glucocorticoids and antibodies are used as immunosuppressive agents to inhibit organ rejection. Calcineurin inhibitors such as cyclosporine A and tacrolimus are most widely used for avoiding organ rejection. In comparison to cyclosporine A, tacrolimus is preferred as it provides a better side effect profile and increased long-term survival in patients.[2,3] In T cells, activation of the T-cell receptor normally increases intracellular calcium, which acts via calmodulin to activate calcineurin. Calcineurin then de-phosphorylates the transcription factor nuclear factor of activated T cells (NF-AT), which moves to

© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 1337–1348

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Tacrolimus-loaded PLGA nanoparticles

Nishita P. Mistry et al.

the nucleus of the T cell and increases the activity of genes coding for interleukin (IL)-2 and related cytokines. Tacrolimus prevents the dephosphorylation of NF-AT.[4] In detail, tacrolimus reduces peptidyl-prolyl isomerase activity by binding to the immunophilin FKBP12 (FK506 binding protein) creating a new complex. This FKBP12-FK506 complex interacts with and inhibits calcineurin, thus inhibiting both T-lymphocyte signal transduction and IL-2 transcription.[5] It is specific T-cell inhibitor, blocks IL 2 production and finally leads to a decrease in T-cell proliferation. It is available as an oral capsule (PROGRAF capsule 0.5 mg, 1 mg and 5 mg) and intravenous injection (PROGRAF inj. 1 mg/ml, 5 mg/5 ml). Dose of tacrolimus depends on the transplanted organ and age of the patient. Tacrolimus shows high pharmacokinetic variability between and within subjects. Due to its narrow therapeutic index, close drug monitoring programmes are required to optimize its efficacy and limit its toxicity.[6] The common adverse events, such as nephrotoxicity, hypertension, diabetogenic effects and neurotoxicity (tremor, seizure and encephalopathy) are frequently observed. This results in discontinuation of tacrolimus immunosuppressive therapy, nephrotoxicity being the dominating reason. High trough levels and augmented concentration of tacrolimus in kidney are the main reasons underlying its nephrotoxic effect. Tacrolimus trough levels 0.05) indicating no effect of galactosylation on the drug release. The in-vitro release data were analysed by different kinetic models to understand drug release mechanism. The regression values of zero order release for Tac-Gal-PLGA NPs were found to be 0.967 and 0.971, respectively. This is supported by Li et al.[29] who reported that the typical release profile of PLGA nanoparticulates is the initial burst followed by zero order kinetics.

In-vivo studies Pharmacokinetic studies

Figure 3 Differential scanning calorimetry thermograms of (a) Tacrolimus (b) physical mixture of tacrolimus and PLGA (c) PLGA (d) physical mixture of tacrolimus and galactosylated PLGA (e) Tacrolimusloaded PLGA nanoparticles and (f) Tacrolimus-loaded galactosylated PLGA nanoparticles.

In-vitro drug release The results of the in-vitro release studies are shown graphically in Figure 6. The release from the plain drug solution was rapid with 85.73% of the drug release within 8 h. However, the drug release from both PLGA and

Figure 7 shows the plasma tacrolimus concentrationtime profiles of plain drug, tacrolimus-loaded PLGA nanoparticles and galactosylated PLGA nanoparticles. The pharmacokinetic parameters are shown in Table 2. The pharmacokinetic studies indicated the concentration of released drug at different time intervals post administration. To selectively estimate the released drug and not the entrapped drug, a solvent (methanol) which could dissolve tacrolimus and not PLGA was selected. From the above observation, it was found that plain drug had higher concentration in plasma at initial time, that is, 735.21 ± 15.21 ng/ml and this eliminated rapidly to 235.07 ± 7.53 ng/ml within 8 h. On the other hand, tacrolimusloaded PLGA nanoparticles and galactosylated PLGA nanoparticles showed lesser tacrolimus concentration in plasma as compared with plain drug at initial time but it increased over 8 h due to sustained release of drug from nanoparticles. Hence, slow increase in concentration of drug in blood with time was observed from nanoparticles. Area under curve (AUC) of plain drug is less compared with nanoparticles formulations, which may be because plain drug got cleared rapidly and encapsulated drug in nanoparticles released for longer period of time (P < 0.05). Thus, the mean residence time of tacrolimus increased upon nanoparticle formulation (Table 2). The volume of distribution (Vd) of plain tacrolimus solution was found to be significantly less than that of nanoparticle formulations. This might be due to the extensive plasma protein binding of free tacrolimus compared with that entrapped in the nanoparticles. There is a significant increase in the half life when tacrolimus is in the nanoparticle form indicating the increased residence time and slow clearance from the body. This is important because when the drug is in systemic circulation, expression of galactose receptors on hepatic tissues may support targeted delivery via receptor mediated endocytosis.


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Figure 4

Transmission electron microscopy images of tacrolimus-loaded (a) PLGA nanoparticles and (b) galactosylated PLGA nanoparticles.

Figure 5

Scanning electron microscopy images of tacrolimus-loaded (a) PLGA nanoparticles and (b) galactosylated PLGA nanoparticles.

Table 2

Results of various parameters of pharmacokinetic study

Parameter

Plain Tacrolimus

Tac-PLGA NP

Tac-Gal-PLGA NP

Cmax (ng/ml) AUC (ng.h/ml) MRT (h) Clearance (ml/h) Half life (h) Volume of distribution (l) Elimination constant (h−1)

735.21 ± 90.40 4272.1 ± 425.34 6.29 ± 0.64 58.51 ± 2.34 4.02 ± 0.46 1.36 ± 0.23 0.172 ± 0.015

523.87 ± 42.32 5059.72 ± 312.45 7.32 ± 0.25 49.40 ± 1.56 33.47 ± 2.56 9.5 ± 1.14 0.0207 ± 0.001

496.64 ± 24.87 4484.54 ± 252.67 7.77 ± 1.74 55.74 ± 3.12 36.28 ± 3.21 11.67 ± 2.14 0.0191 ± 0.004

AUC, Area under curve; MRT, Mean residence time; PLGA, poly lactic-co-glycolic acid; Tac-Gal-PLGA NP, tacrolimus-loaded galactosylated PLGA nanoparticle; Tac-PLGA NP, tacrolimus-loaded PLGA nanoparticle .

Biodistribution studies The results of biodistribution studies are shown in Table 3. Biodistribution studies were performed to assess the targeting ability of galactosylated nanoparticles to deliver tacrolimus to the hepatic tissues. The concentration of tacrolimus in liver was significantly higher in nanoparticle formulations than plain drug, which may be due to uptake of nanoparticles by mono-nuclear phagocytic system.[11] However, the concentration in nanoparticles was significantly less than galactosylated PLGA nanoparticles (P < 0.05) that might be due to non-specific entry in 1344

the absence of specific receptors. At 24 h, there was an increase in tacrolimus concentration from Tac-GalPLGA NPs by 4.83 fold and 11.72 fold from Tac-PLGA NPs and plain drug solution respectively. In case of Tac-PLGA NPs, galactosylation facilitated the receptor mediated endocytosis via Asialoglycoprotein receptor (present on liver cells only) on liver cells which facilitated selective entry in liver[12] This illustrates the preferential targeting of nanoparticles to liver tissue followed by a sustained release in the vicinity, augmented bioavailability and elevated retention potential of the formulation in liver. In spleen, tacrolimus concentration were higher by 4–5-fold at 1 h, 8 h




Figure 6 % Cumulative tacrolimus release from plain drug solution, tacrolimus loaded PLGA nanoparticles and tacrolimus-loaded galactosylated PLGA nanoparticles.

Figure 7 Mean plasma tacrolimus concentration versus time curves obtained after i.v. administration of plain drug solution, tacrolimus loaded PLGA nanoparticles and tacrolimus-loaded galactosylated PLGA nanoparticles.

and 24 h in Tac-Gal-PLGA NPs (P < 0.05) in comparison to plain drug solution which might be due to mononuclear phagocytic uptake of nanoparticles by liver and spleen. Presence of high concentration of tacrolimus in liver and

spleen will increase its uptake by the T cells resulting in inhibition of production of dephosphorylated NF-AT which initiates the immune response. Thus, availability of higher concentration of tacrolimus at the site of action can


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Table 3 Results of biodistribution studies in liver, spleen and kidney upon administration of plain tacrolimus solution, tacrolimus-loaded PLGA and galactosylated PLGA nanoparticles Time (h) Liver 1 8 24 Spleen 1 8 24 Kidney 1 8 24

Concentration of tacrolimus from plain tacrolimus solution (ng/g of organ)

Concentration of tacrolimus from Tac-PLGA NPs (ng/ g of organ)

Concentration of tacrolimus from Tac-Gal-PLGA NPs (ng/g of organ)

970.40 ± 12.43 947.12 ± 19.21 352.75 ± 18.93

1233.30 ± 129.2 1622.67 ± 98.46 4136.59 ± 138.8

9 575.19 ± 197.4 17 774.11 ± 158.4 19 995.21 ± 153.2

412.13 ± 71.19 358.7 ± 59.73 106.06 ± 39.71

1739.31 ± 98.27 1497.61 ± 42.34 352.75 ± 102.2

1 685.90 ± 69.93 1 285.45 ± 84.61 52 448 ± 79.26

468.78 ± 28.3 347.24 ± 38.6 105 ± 17.9

98.27 ± 18.6 109.3 ± 12.8 53.2 ± 8.3

55.83 ± 12.1 75.32 ± 17.32 31.79 ± 8.7

PLGA, poly lactic-co-glycolic acid; Tac-Gal-PLGA NP: tacrolimus-loaded galactosylated PLGA nanoparticles; Tac-PLGA NP: tacrolimus-loaded PLGA nanoparticles.

Table 4

Effect of storage condition on particle size and drug retained in tacrolimus-loaded nanoparticles Initial

Storage condition Particle size (nm) Refrigeration (5°C ± 3°C) Room temperature (25°C ± 5°C) Zeta potential (mV) Refrigeration (5°C ± 3°C) Room temperature (25°C ± 5°C) % drug retained (%) Refrigeration (5°C ± 3°C) Room temperature (25°C ± 5°C)

After 1 month

Tac-PLGA NPs

Tac-Gal-PLGA NPs

Tac-PLGA NPs

Tac-Gal-PLGA NPs

129.5 ± 0.92 129.5 ± 0.92

132.3 ± 1.03 132.3 ± 1.03

132.5 ± 1.21 173.6 ± 1.28

135.3 ± 1.17 182.3 ± 0.78

−37.3 ± 2.34 −37.3 ± 2.34

−25.3 ± 2.16 −25.3 ± 2.16

−36.45 ± 2.42 −44.23 ± 3.12

−36.32 ± 1.12 −43.28 ± 3.73

84.38 ± 1.64 84.38 ± 1.64

83.45 ± 1.75 83.45 ± 1.75

83.12 ± 1.29 55.21 ± 1.32

82.36 ± 1.14 58.32 ± 0.93

PLGA, poly lactic-co-glycolic acid; Tac-Gal-PLGA NP, tacrolimus-loaded galactosylated PLGA nanoparticle; Tac-PLGA NP, tacrolimus-loaded PLGA nanoparticles.

lead to its enhanced immunosuppressive effect leading to increased chances of graft survival. This is supported by work done by Afifi et al.[30] who prepared poly (lactic acid) nanoparticles of tacrolimus for liver targeting. In kidney, it was observed that tacrolimus concentration was significantly higher (P < 0.05) in plain drug as compared with nanoparticle formulations. Also, galactosylated nanoparticles showed significant reduction in tacrolimus concentration in kidney as compared with nongalactosylated PLGA nanoparticles. From Tac-Gal-PLGA NPs, there was decrease in tacrolimus concentration by 8.3-, 4.6- and 3.3-fold at 1 h, 8 h and 24 h, respectively, in comparison to plain drug solution. It indicates that nephrotoxicity of tacrolimus was minimized upon administration of Tac-Gal-PLGA NPs. The biodistribution studies thus proved the site specificity of the nanoparticles to liver. Enhanced liver concentrations may prove to be hepatotoxic. However, dose adjustment is possible in targeted drug delivery systems, which have the potential of producing the 1346

desired effects at lower concentrations because of the sitespecific accumulation. This will consequently lower the dose-related side effects. Detailed toxicity studies may be able to provide data about the concentration which causes liver toxicity.

Stability studies It was observed that there was comparatively negligible change (P > 0.05) in particle size, zeta potential and % drug retained of Tac-Gal-PLGA NP formulation (Table 4) at refrigeration condition (5°C ± 3°C) as compared with room temperature (25°C ± 5°C). Thus, nanoparticle formulations should be stored at refrigeration condition (5°C ± 3°C). On storage at room temperature % drug retained decreased, it might be due to drug leakage from nanoparticles. Particle size also increased during that time which could be due to particle aggregation which is known as Ostwald ripening (the particle aggregate with each other and forms larger




particle). The zeta potential remained same on storage at refrigerated condition indicated that the galactosylation has retained on storage.

Conclusion The present study was aimed to provide targeted delivery of tacrolimus to liver. Galactosylated PLGA nanoparticle seems to be a promising carrier for targeting to liver and thereby reduced side effects of drug. The uptake of prepared galactosylated PLGA nanoparticles by liver was significantly higher than that of the plain drug solution and plain PLGA nanoparticles. Galactosylation facilitated receptor mediated endocytosis via asialoglycoprotein receptor on liver cells which resulted into selective entry in liver. Similar fashion was observed in spleen while the concentration in kidney

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Declarations Conflict of interest The authors report no declaration of interest.

Acknowledgement Authors acknowledge the TIFAC CORE in NDDS, Government of India, New Delhi for providing the research facilities to the team.

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