Design of Mannose-Coated Rifampicin nanoparticles modulating the immune response and Rifampicin induced hepatotoxicity with improved oral drug delivery

2.1 Materials

Chitosan (Cht) (low molecular weight with degree of acetylation 75–85%), thioglycolic acid (TGA), D-mannose, sodium borohydride and sodium tripolyposhphate (TPP), were obtained from Sigma Aldrich (Germany) while nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), hydroxylamine, sodium cyanborohydride, 1-ethyl-3–3(3-dimethyl aminopropyl carbodiimide hydrochloride (EDAC), mucin, verapamil, thiobarbituric acid, triazole, chloroform and Ellman’s reagent were purchased from Merck (USA). Rif was given as a gift from Pfizer. All the solvents used in the experimentation were of HPLC and analytical grade. Chemicals and reagents were of analytical grade and were used as received.

2.2 Synthesis and basic characterization of nanoparticles

The synthesis MPTCht-NPs loaded with Rif has been reported in our previous work (Rauf et al. 2020). Briefly, these NPs were synthesized by ionic gelation method. For polymer enveloped NPs preparation, the conjugated polymer solutions (0.2% w/v) were prepared. The Cht solution was prepared in 1% (v/v) aqueous acetic acid (pH 4) while the solutions of TCht, PTCht and MPTCht were prepared in deionized water. The Rif (1 mg/ml) was dissolved in DMSO and diluted with PBS (pH 4). The TPP (0.2% w/v) was prepared to which the Rif solution was added and this mixture is then added dropwise into the polymer solution with continuous stirring until the formation of milky suspension. The resultant suspension was then centrifuged at 13,500 rpm for 30 min and the formed palette of NPs were then collected and redispersed in 3% trehalose solution, lyophilized and then stored at 4 °C until further use. The NPs were characterized for particle size (nm), zeta potential (mV), polydispersity index (PDI) using Nano-zeta sizer (Malveern, UK). The morphological studies were analyzed via scanning electron microscopy (SEM) (FEI Nova NanoSEM 450, USA). The Encapsulation efficiency (EE%) was also measured via High pressure liquid chromatography (HPLC) by indirect method by using the following formula: $$EE\left(\%\right)=Amountofdruginformulation/totalamountofdrugadded\times 100\%$$

The drug loading (DL) was also calculated by the following formula: $$\mathrm{D}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\%=\frac{\mathit{Weight}ofdrugused-amountofunbounddrug}{\mathit{Amount}ofnanoparticlesrecoverd}\times 100$$

2.3 Swelling studies and mucoadhesion

The swelling behavior of Cht, TCht, PTCht and MPTCht-NP were carried out to evaluate the mucoadhesion properties. Weighed quantity (30 mg) of each sample was taken and formulated as tablet (5 mm) by direct compression. The tablets were fixed with the tip of the needle and immersed in the phosphate buffer saline (0.1 M PBS 25 ml) of pH 7.0 maintained at temperature of 37 ℃. At different time points, the tablets were taken out and excess water was absorbed by tissue paper. Tablet was weighed again to estimate the amount of water absorbed by the following formula: $$\mathit{Swelling}index\left(\%\right)=\left(\mathit{Wt}-Wo\right)/Wo\times 100$$

Here, W₀ is the weight of dry tablet; Wt is the final weight after specific time interval.

For mucoadhesion, 4 g of mucin dissolved in PBS (pH 7.4, 50 ml). The pH was then adjusted to 6.8. Then the final NPs were dispersed in distilled water (50 w/v). NP solution was mixed with equal volume of mucin solution (pH 7.5 with 0.1 M PBS). After 15–20 min, the minute amount of the above mixture was placed on the cone plate viscometer (AMETEK Brookfield) and equilibrated for almost 3 min (37 ± 2 ℃). Apparent viscosity as well as G' (storage modulus) and G“ (loss modulus) were observed as previously reported (Sarwar et al. 2018).

2.4 Ex-vivo permeation studies

All animal experiments were approved by the local ethical committee i.e., from bioethical committee Quaid-i-Azam University, Islamabad (BEC-FBS-QAU2019-202). The ex vivo permeation study of Rif in comparison with prepared NPs was carried out by using everted sac method. This study was performed on the intestine of healthy rats (200–300 g). The animals were sacrificed by the cervical dislocation method to give minimum possible pain of death. The intestines of the sacrificed rats were removed and washed with Kreb’s ringer solution. After washing, the intestine was cut into small segments of 4–5 cm. For everted sac, narrow glass rod was passed through one open end of the intestinal segment and then gently rolling down the segment over it. To enhance the wettability of Rif, tween-80 was added. One end of the intestinal segment was tied and sample is filled by the dispersed solution (3 ml) of prepared NPs containing 2.7 mg Rif using the syringe needle, and the other end tied in the same manner in order to make a close sac. Verapamil, a P-gp inhibitor, was filled in one sac (100 µg/ml) to compare the Papp enhancement with prepared NPs. These filled intestinal segments were then immersed in the beakers containing 10 ml of the Krebs’s solution at 37 °C in water bath shaker under continuous stirring. At definite time intervals, the samples were collected from the surrounding medium and replaced with the equal amount of fresh medium accordingly (Afzal et al. 2019). The collected samples were then analyzed for quantification by HPLC using C-18 column. The optimized mobile phase consists of mixture of phosphate buffer (pH 6.5) and acetonitrile (6:4) with methanol as diluent. The prepared solution was passed through 0.45 µm filter and degassed by sonication of 30 min. The flow rate was maintained at 1 ml/min at 280 nm and samples were analyzed at room temperature. To extract Rif, 500 µl of methanol was added to 200 µl plasma sample. The sample was then vortexed and then centrifuged at 10,000 rpm for 15 min. Afterwards, 20 µl of supernatant was taken and analyzed by HPLC (Rauf et al. 2021).

Papp was calculated as follows: $$\mathit{Papp}\left(\mu g/c{m}^2\right)=\left(\mathit{Concentration}\times Volume\right)/\left(\mathit{MSA}\left(\mathit{mu}cosalsurfacearea\right)\right)$$ $$\mathit{Where}MSA\left(\mathit{mu}cosalsurfacearea\right)=C\left(\pi \times diameter\right)\times Length$$

2.5 Hemolysis study

For in vitro hemolysis assay, fresh human blood was taken and washed thrice with Dulbecco PBS. After each washing at 150 rpm for 5 min, the red blood cells (RBCs) were pelleted out while supernatant was discarded. The final pellet was 9 times diluted (v/v) with PBS. Afterwards, the RBCs were seeded into 96-well plate and treated with different concentrations of Rif and NPs. Empty NPs served as negative control while triton x served as positive control to estimate the hemolysis induced by the NPs. After incubation of 24 h, the absorbance was measured by microplate reader (Perkin-Elmer, USA) at 570 nm. Hemolysis % and RBCs viability was calculated by using following formula (Sarwar et al. 2018):

$\mathbf{H}\mathbf{e}\mathbf{m}\mathbf{o}\mathbf{l}\mathbf{y}\mathbf{s}\mathbf{i}\mathbf{s}\%=(\frac{\mathit{S}\mathit{a}\mathit{m}\mathit{p}\mathit{l}\mathit{e}\mathit{a}\mathit{b}\mathit{s}\mathit{o}\mathit{r}\mathit{b}\mathit{a}\mathit{n}\mathit{c}\mathit{e}-\mathit{N}\mathit{P}\mathit{s}\mathit{a}\mathit{b}\mathit{s}\mathit{o}\mathit{r}\mathit{b}\mathit{a}\mathit{c}\mathit{n}\mathit{e}}{\mathit{A}\mathit{b}\mathit{s}\mathit{o}\mathit{r}\mathit{b}\mathit{a}\mathit{n}\mathit{c}\mathit{e}\mathit{o}\mathit{f}\mathit{P}\mathit{C}-\mathit{A}\mathit{b}\mathit{s}\mathit{o}\mathit{r}\mathit{b}\mathit{a}\mathit{n}\mathit{c}\mathit{e}\mathit{o}\mathit{f}\mathit{N}\mathit{C}})$  × 100 $$\mathbf{R}\mathbf{B}\mathbf{C}\mathbf{s}\mathbf{V}\mathbf{i}\mathbf{a}\mathbf{b}\mathbf{i}\mathbf{l}\mathbf{i}\mathbf{t}\mathbf{y}\%=100-(\frac{\mathit{S}\mathit{a}\mathit{m}\mathit{p}\mathit{l}\mathit{e}\mathit{a}\mathit{b}\mathit{s}\mathit{o}\mathit{r}\mathit{b}\mathit{a}\mathit{n}\mathit{c}\mathit{e}-\mathit{N}\mathit{P}\mathit{s}\mathit{a}\mathit{b}\mathit{s}\mathit{o}\mathit{r}\mathit{b}\mathit{a}\mathit{c}\mathit{n}\mathit{e}}{\mathit{A}\mathit{b}\mathit{s}\mathit{o}\mathit{r}\mathit{b}\mathit{a}\mathit{n}\mathit{c}\mathit{e}\mathit{o}\mathit{f}\mathit{P}\mathit{C}-\mathit{A}\mathit{b}\mathit{s}\mathit{o}\mathit{r}\mathit{b}\mathit{a}\mathit{n}\mathit{c}\mathit{e}\mathit{o}\mathit{f}\mathit{N}\mathit{C}}\times 100)$$

Where, NC = negative control, PC = positive control.

2.6 In vivo pharmacokinetics and biodistribution

The female rabbits (2000 ± 150 g) were obtained from animal house and maintained in air-conditioned room at 25 ± 2 °C with 12 h dark and 12 h light cycle with water and food provided ad libitum. Animals were divided into 6 groups of 5 animals in each group. Group 1 was given normal saline and was considered as control while group 2 was given Rif suspension at a dose of 12 mg/kg. Group 3, 4, 5 and 6 were administered Cht-NP, TCht-NP, PTCht-NP and MPTCht-NPs respectively through oral gavage. Blood samples were collected by jugular vein at various time points in heparinized microcentrifuge vials. The blood samples were then centrifuged at 10,000 rpm for 15 min and plasma was separated. After predetermined time intervals, the animals were sacrificed by placing the animals in the anesthetic chamber with a cotton soaked in halothane and vapors were inhaled. After sacrifice, all vital organs such as kidneys, lungs, liver and spleen were removed and their homogenates were prepared. Drug concentration in both plasma and organs homogenates was determined by HPLC following standard protocols (Sarwar et al. 2018).

2.7 Acute oral toxicity studies and hepatotoxicity modulation

The acute oral toxicity of the prepared NPs was evaluated for 14 days in female Wistar rats following the OECD guidelines. Healthy female Wistar rats (80 ± 12 g) were obtained from animal house and were kept under controlled conditions of temperature with food and water supplies. The animals were grouped into 6 groups having 5 animals in each group. Group 1 was administered normal saline and served as control. Group 2 was administered Rif suspension while groups 3, 4, 5 and 6 were administered Cht, TCht, PTCht and MPTCht NPs, respectively. The dose of 12 mg/kg was given through oral gavage. The rats were kept under observation for 24 h for any changes in weight and behavioral pattern, skin, fur, feces, urine, salivation, respiration and sleep pattern. The animals were also observed for any visual mortality, change in physical appearance and signs of any illness throughout the week. After 14 days, the blood was collected for hematology and serum analysis. Animals were then sacrificed by cervical dislocation and all vital organs were removed for tissue histological examination (Sohail et al. 2017).

2.8 Immunomodulation and hepatotoxicity modulation (Bax and Bcl-2 expression) by RT-PCR

For immunomodulation and hepatotoxic modulation potential of prepared NPs of Rif, the standard protocol of RT-PCR was used with slight modifications.

3.1 Basic characterization, swelling and mucoadhesion

The prepared MPTCht-NPs were 300 ± 20 nm in size with 73 ± 4% EE and 18 ± 4 zeta potential (Fig. 1). The detail of the NPs synthesis, composition, size, zeta potential, EE%, and DL % is given in Table 1. The swelling capacity of NPs influences the mucoadhesion with mucosa or skin, drug release and stability. Once attached with the mucus layer, NPs absorb water through capillary action from the underlying mucus membrane thus swelling up and developing mucoadhesion while initiating drug release. The mucoadhesion; however, should be in optimal limits to control the drug release for sufficient period of time. As such, the immediate abrupt swelling develops weak adhesion while slow swelling develops localized mucosa related issues while retarding drug release (Afzal et al. 2019). Therefore, the swelling of the polymers Cht, TCht, PTCht and MPTCht was performed and results are presented in Fig. 2A. While the PTCht showed slow and gradual higher swelling compared to other polymeric tablets. The swelling of the MPTCht-NPs tablet showed better water uptake showing slow and gradual uptake as compared to other polymeric tablets. MPTCht showed decreased swelling which may be attributed to the presence of surface mannose groups. These surface groups may result in decreased water uptake resulting in lower swelling index compared to Cht, TCht and PTCht. This swelling behavior affects the drug release from formulations in a controlled manner.

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Fig. 2

A) Swelling studies of Cht, TCht, PTCht and MPTCht NPs. B) Permeation enhancement of Rif from Rif + Ver, Cht, TCht, PTCht and MPTCht from apical to basolateral and C) basolateral to apical membrane of rat intestine. D) In vitro hemolysis assay performed on fresh human red cells. All experiments were performed in triplicate and results are shown as mean ± SD and statistically significant differences were evaluated by one-way ANOVA followed by Dunnhet’s multiple comparisons test at significance level of *p < 0.05, **p < 0.01 and ***p < 0.001 vs control.

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Mucoadhesion property of the NPs is an important consideration for oral drug delivery. The mucoadhesion property of the polymer system prolongs the contact time with the mucus membrane resulting in sustained drug delivery with reduced dosage frequency (Soane et al., 1999). Different studies demonstrated the drug combination with Cht resulted in increased contact time of this system with the mucus. This increased contact time with mucosa is due to the inbred capacity of its protonated amino groups to interact with the mucus. Mucus is composed of the glycoprotein mucin having a negative charge due to the presence of sialic acid residues. The intent of mucoadhesion depends on the deacetylation degree of Cht and the amount of sialic acid residues. The process of adhesion is complex involving the steps of contact, formation and consolidation of some bond between the polymer and the mucus layer (He et al., 1998). The thiolation of the Cht results in the formation of thiolated Cht which forms the disulfide bonds with the mucin glycoprotein. Further preactivation of these thiomers will protect them from oxidation so more active thiol groups will be available to interact with mucus layer (Bernkop-Schnurch, 2011). The process of mucoadhesion of polymeric drug delivery system involves 3 steps: 1) preliminary swelling which results in initiation of contact of the polymeric system with biological tissues; 2) interpenetration and entanglement of mucoadhesive polymeric chains with mucin chains; and 3) formation of chemical bonds between the entangled chains. The mucoadhesive NPs remain attached to the mucosa for relatively longer period of time providing increase contact time resulting in better absorption of NPs. Increased contact time with mucosa also provides the continuous pool of drug for extended time period in order to give better drug absorption (Afzal et al., 2019). The mucoadhesion of the prepared TCht, PTCht and MPTCht NPs was assessed by the rheological synergism. Mucus contains free thiol groups which will form the disulfide linkage with the thiomers. In this regard, the viscoelastic behaviors of the completely hydrated Cht, TCht, PTCht, MPTCht and mucin may reflect the mucoadhesive strength of the polymers. The higher the rheological synergism, the stronger the interaction with mucus layer, which results in better gastric absorption of NPs. The viscoelastic parameters were measured as presented in Table 2. The values of G and G’ for TCht, PTCht and MPTCht with mucin were higher compared to unmodified polymer i.e., Cht. This increase may be due to the formation of S-S of thiol with the mucin. Mucin is rich in cysteine subunits having -SH groups. The thiomers have -SH groups that interact by forming disulfide exchange reaction by forming S-S thus forming covalent bond with mucus resulting in strong mucoadhesion strength. The considerable lack of viscoelastic parameters of Cht is due to the absence of thiol groups in the polymer backbone. These findings clearly demonstrate the advantage of using the thiomers for purpose of oral absorption of NPs (Afzal et al. 2019).

3.2 Ex vivo permeation and hemolysis study

One of the objectives of the present study was to enhance the permeability of the Rif loaded NPs. The efflux transporters present on the intestinal epithelial membrane, for instance P-gp have considerable influence on drug absorption. Besides P-gp, paracellular and transcellular pathway also significantly affect the absorption and oral bioavailability of drugs. The paracellular route is guarded by two proteins occludin and claudin (responsible for cell adhesion and ion selection) that can be opened reversibly by different strategies. Thiomers and preactivated thiomers (second generation thiomers) have the capability of reversible opening of these junctions. Also, the thiomers have the intrinsic property of P-gp inhibition while increasing the drug transport across the intestine by keeping the P-gp inhibited. The absorption of Rif was evaluated alone and in the presence of P-gp inhibitor i.e., verapamil. The results of the Rif in the presence and absence of verapamil on the everted and non-everted rat intestine are presented in the Fig. 2 (B, C). Our results demonstrated that in the presence of verapamil, the transport of Rif was slightly increased compared to control buffer, suggesting that Rif is the substrate for P-gp. The Papp of Rif solution across rat mucosa was 80 folds that of absorptive (apical to basolateral) suggesting that the movement of drug is secretory oriented. The presence of efflux pump on the apical surface suggests that the secretory transport of the Rif occurred through transcellular route. The Papp enhancement values of Cht-NPs was 8 folds higher than Rif solution while TCht, PTCht and MPTCht NPs showed 11-, 16- and 19-fold high enhancement in comparison to pure Rif (p < 0.05). The results (Table 3) suggest the possible opening of paracellular pathway by the thiomers which improves the transport of Rif. The preactivated thiomers further increase the transport because they have stronger effect on loosening of tight junctions because they directly affect the integrity of tight junction thereby increasing drug uptake (Bernkop-Schnürch et al. 2003). The opening of tight junctions is associated with the interaction of thiomers with the desmosomes of tight junctions by disulfide linkage and reversibly opens them. The permeation improvement by inhibition of protein tyrosine phosphatase (PTP) occurs by the S-S formation of thiomers with the cysteine site of protein. Consequently, this binding results in higher degree of phosphorylation of protein contributing the opening of tight junctions. Hence, appreciably improved permeation across the tight junctions was examined (Sohail et al. 2016).

The hemolysis assay detailed insight about the biocompatibility of the nano formulations for in vivo application. The detail of the viable RBCs cells after treatment with different nano formulations is given in the Fig. 2D. The Cht-NPs exhibited the viability of 85 to 60 % at the concentration of 10 to 200 µg/ml while MPTCht-NPs exhibited the viability of 99 to 80 % at the concentration of 10 to 200 µg/ml. The RBCs viability with MPTCht nano formulation was greater than the Rif and other nano formulations which indicated the biocompatible nature of the MPTCht-NPs.

3.3 Acute oral toxicity

The in vivo acute oral toxicity of the prepared NPs at the dose of 12 mg/kg was observed in Wistar female rats. The rats showed no sign of any change in behavior, fur, skin, and bowel habits during first day that prevailed throughout the week. Also, no mortality and weight change were observed throughout the study. After 14 days, the blood was collected by cardiac puncture in heparinized tubes and animals were sacrificed in order to collect vital organs for further analyses.

3.3.1

3.3.1 Hematology and serum biochemistry

The biocompatibility of NPs is very critical once they come in contact with blood and organs because inflammatory response is most likely to occur depending on the level of incompatibility. Complete blood count (CBC) was performed in order to evaluate the biocompatibility of the prepared nano formulations with blood as shown in Table 4. The results indicated decreased Hb level and RBC count, increased hemolytic behavior of Rif and Ch-NP, while the hemolytic behavior of the MPTCht-NP was negligible as compared to Rif. The level of white blood cells (WBCs) was not affected by the treatment with NPs. Other parameters such as MCH, MCV, PCV, PDW% were also analyzed.

Table 4 Blood parameters.

Blood Blood parameters	ContControl	Rif	Cht	TCht	PTCht	MPTCht
RBC (1012/L)	6.98 ± 1.22	4.83 ± 1.09	6.34 ± 2.98	7.1 ± 4.9	5.01 ± 3.60	6.94 ± 2.70
MCV(fL)	62.5 ± 2.59	60.5 ± 3.88	55.8 ± 3.78	58.8 ± 4.7	63 ± 3.66	57.9 ± 3.84
MCH (pg)	21.3 ± 4.76	20.4 ± 1.55	21.3 ± 4.5	20.8 ± 3.44	22.3 ± 2.66	22.5 ± 2.75
PCV (%)	36.4 ± 4.87	36.4 ± 0.90	31 ± 3.8	33.7 ± 2.78	39.9 ± 3.99	30.1 ± 3.65
Hb (g/dL)	14.9 ± 2.34	10.1 ± 0.98	10.3 ± 2.7	14.8 ± 2.33	11.2 ± 2.90	14.3 ± 2.65
WBC (109/L)	5.5 ± 0.56	5.1 ± 0.87	6.1 ± 1.34	8.1 ± 0.65	15 ± 3.46	6.4 ± 1.98
Platelets 109/L	963 ± 7.68	656 ± 5.66	64 ± 3.99	774 ± 4.77	699 ± 5.66	57 ± 2.78
RDW (%)	17.5 ± 1.45	16.7 ± 3.44	15.9 ± 2.67	16.6 ± 3.23	19.8 ± 0.89	13.6 ± 1.88
MPV (fL)	6.9 ± 2.33	7.8 ± 3.6	6.9 ± 0.87	7.1 ± 1.54	9.1 ± 0.88	7.1 ± 1.87

After 14 days treatment, the effect of NPs on serum biochemistry was also analyzed. LFTs shown in Fig. 3A suggest no effect on albumin levels after treatment. The level of serum transaminases such as AST, ACT AND ALP in Rif treated rats was higher compared to other groups while rats treated with MPTCht-NPs did not increase transaminasesmas shown in the Fig. 3A. The transaminase levels depict the cellular integrity level of the liver. Higher level of SGPT, SGOT and ALP indicates necrosis or cellular damage resulting in extravascular leakage of these enzymes. Rif showed higher levels of these enzymes while all NPs showed normal and acceptable levels of these enzymes (Sohail et al. 2016). There was no change in bilirubin level with any treatment group. The effect of nano formulations on kidneys was assessed through RFTs and the results showed no significance difference from the normal reference values (Fig. 3B). Creatinine level also remained unaffected while BUN level was increased with Cht-NP while no significant change was observed with MPTCht-NP compared to control. The levels of liver stress markers MDA increased in rats treated with Rif in comparison to control and MPTCht-NPs (Fig. 3C). While the level of antioxidant GSH in Rif loaded MPTCht-NPs were enhanced compared to free drug Rif treated and control group (Fig. 3D). The hepatotoxicity associated with Rif is related to excessive reactive oxygen species (ROS) production and mitochondrial fragmentation leading to hepatic cell membrane damage. The results of serum transmaninases and oxidative stress markers revealed the hepatoprotrctive nature of Rif loaded MPTCht-NPs via sustained release of the drug.

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Fig. 3

A) LFTs B) RFTs performed on rat serum after 14 days of acute oral toxicity studies. Effect of oral administration of Rif and MPTCht-NPs after 14 days on C) liver MDA D) and GSH levels. E) Organ to body ratio of different treatments evaluated on liver, lungs, spleen and kidneys. All experiments were performed in triplicate and results are shown as mean ± SD and statistically significant differences were evaluated by one-way ANOVA (**p < 0.05, **p < 0.01 and ***p < 0.001 vs control).

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3.3.3

3.3.3 Tissue histology of vital organs

Along with serum biochemical analysis, the tissue histological studies were performed. The stained slides were examined microscopically for any visual signs of toxicity of the NPs. No change in kidney and lungs cellular morphology was observed in all treatments supporting biocompatibility of the NPs (Fig. 4A). Rif treated group exhibited considerable liver damage including liver swelling, binucleation with enlarged nucleus, degeneration, central vein congestion, apoptosis and necrosis. The liver sections of rats treated with Rif loaded MPTCht-NPs showed no significant signs of liver toxicity or abnormality as compared to Rif treated group (Fig. 4B).

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Fig. 4

A) Microscopic examination of tissue histology slides of organs of different treatment groups i.e., Control, Rif, Cht, TCht, PTCht and MPTCht-NPs (x400). B) Microscopic examination of liver (x400) from a) control group with normal tissue histology b) Hydropic degeneration of Rif treated liver c) Lobular hepatitis d) apoptosis e) Vein congestion f) Portal tract expansion g) Rif treated liver morphology h) Empty NPs treated group showing normal histology and i) MPTCht-NPs group with no obvious abnormality of liver tissue.

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3.4 Quantitative estimation of immune cells, Bcl-2 and Bax gene

The immunomodulation properties of MPTCht-NPs were evaluated by RT-PCR. Macrophages are the major effector cells that are thought to be involved in the pathogen eradication. The appropriate activation of these effector cells is therefore necessary. Th-2 cytokines i.e., IL-6 and IL-10 play a role in reducing Th-1 cytokines i.e., TNF-α and IL-12 thereby causing the pathogen dissemination and reduction in anti-tubercular activity. Effective ATD therapy is associated with increased Th-1 cytokines production which is involved in the enhanced host immunity. Many cytokines are produced in response to the infection with mycobacterium (Afzal et al. 2019). The levels of Th-1 cytokines i.e., IL-12 was enhanced after treatment with MPTCht-NPs while no significant difference was observed in production of IL-10 and IL-6 in comparison to unmodified Cht as shown in Fig. 5 A, B and C. The apoptosis is regulated by the caspase family of proteins. There are 3 types of caspases that are thought to be involved in apoptosis: (1) caspases 2, 9 and 10 are initiator caspases that initiate the apoptosis signal; (2) caspases 3, 6 and 7 are executioner caspases that carry out mass proteolysis that led to apoptosis; (3) and caspases 1, 4, 5, 11 and 12 are inflammatory caspases that are not directly involved in apoptosis rather they are involved in pyroptosis (Brentnall et al. 2013). The initiator caspases activate the executioner caspases that will lead to the proteolysis of the structural proteins leading to apoptosis of the infected cells via extrinsic or intrinsic pathway. We explored the extrinsic pathway of induction of caspase 9 which in turn leads to the activation of intrinsic pathway of apoptosis by the initiation of endoplasmic reticulum (ER) stress. The RT-PCR data suggest increase in the expression level of initiator caspase 9 which in turn leads to the activation of executioner caspases 3 and 7 after treatment with coated MPTCTh-NPs, which in turn would increase the apoptosis of M.tb infected cells (Fig. 5 D,E and F) (Mcllwain, 2013; Momoi, 2004; Winter, 2014) (Fig. 4 D, E and F).

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Fig. 5

Gene expression analysis of cytokines and caspases produced by infected macrophages via RT-PCR A) IL-6 expression analysis B) IL-10 expression analysis C) IL-12 expression analysis D) Caspase-3 E) Caspase-7 and F) Caspase 9 expression analysis of macrophages infected with mycobacterium and treated with NPs. G) Gene expression levels of anti-apoptotic (Bcl-2) and pro-apoptotic gene (Bax) between different experimental groups. All experiments were done in triplicate and results are shown as mean ± SD and statistically significant differences were evaluated by one-way ANOVA (*p < 0.05, **p < 0.01 and ***p < 0.001 vs control).

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Bcl-2 and Bax levels are associated with mitochondrial-associated cellular apoptosis. The levels of Bcl-2 (anti-apoptotic protein) decreases while levels of Bax (pro-apoptotic protein) increases in apoptosis. The gene expression level of Bcl-2 in Rif treated group was significantly reduced in comparison to the Rif loaded MPTCht-NPs and control. While the level of Bax gene was significantly increased in Rif treated group in comparison to control and Rif loaded MPTCht-NPs treated group. These results strengthened the possible hepatoprotective effect of MPTCh-NPs as shown in Fig. 5G.

3.5 In vivo pharmacokinetics and biodistribution studies

Rif is well absorbed orally from the gastrointestinal tract but it suffers the variable bioavailability issue due to altered gastric pH and delayed gastric emptying time. The in vivo pharmacokinetics and biodistribution studies were performed on healthy rabbits and the plasma and organ drug levels were estimated by HPLC. Various pharmacokinetic parameters of Rif and NPs (Cht, TCht, PTCht and MPTCht) are given in Table 5. The in vivo pharmacokinetic data clearly revealed the altered behavior of drug with improved properties when administered through mannose-conjugation (MPTCht). The MPTCht and PTCht NPs showed Cmax of 18.65 µg/ml and 11.1 µg/ml, which is significantly higher than Rif i.e., 2.5 µg/ml. The t_1/2 of MPTCht and PTCht NPs was significantly higher as compared to Rif. Moreover, significant plasma levels of MPTCht-NPs were observed after 3 days but the levels of Rif were only observed till 24 h. Table 5 clearly indicates that the clearance of the MPTCht was delayed in comparison to free drug which is rapidly cleared from the blood and detected only till 24 h. The delayed clearance of MPTCht-NPs exhibited the prolonged circulation and higher distribution of these NPs in plasma. Since Rif is bound inside the polymeric matrix in MPTCht-NPs and will be protected from its own metabolism so only free drug will be eliminated after metabolic conversion. Increase in oral bioavailability of 16 folds was observed with MPTCht as compared to Rif. The enhanced bioavailability of the MPTCht was attributed to the combined effects of swelling, mucoadhesion and increased paracelullar transportation that is possibly attributed to thiol groups. The plasma drug concentrations of all NPs are depicted in Fig. 6A.

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Fig. 6

A) Plasma drug concentration of Rif after oral administration of Rif suspension, Cht, TCht, PTCht and MPTCht NPs at oral dose of 12 mg/kg. All blood samples were collected at predefined time intervals till 72 h and analyzed via HPLC for Rif quantification. B) Percentage drug accumulation in different organs (liver, spleen, kidney and lungs) after treatment with Rif and C) MPTCht-NPs.

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The in vivo biodistribution studies were performed in order to investigate the target specificity of the MPTCht NPs. The % drug accumulation of Rif in all vital organs was quantified by HPLC as presented in Fig. 6B, C. The results of biodistribution of MPTCht clearly indicated that the macrophage rich organs (liver, spleen and lungs) accumulated significantly higher Rif concentration as compared to free drug Rif. The higher uptake of Rif into liver, spleen and lungs was owing to the receptor mediated uptake of mannose-conjugated NPs (MPTCht) by the macrophages.

Institutional Review Board Statement

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Informed Consent Statement

Not applicable.