Targeting kidneys by superparamagnetic allopurinol loaded chitosan coated nanoparticles for the treatment of hyperuricemic nephrolithiasis

Abstract

Purpose

The major short coming of conventional therapy system is that they can’t deliver the therapeutics specifically to a site within the body without producing nonspecific toxicity. Present research aimed at developing kidney targeted allopurinol (AP) loaded chitosan coated magnetic nanoparticles (A-MNPs) for the management of hyperuricemic nephropathy manifested in the form of nephrolithiasis.

Methods

The work includes preparation of magnetic nanoparticles by chemical co-precipitation method and evaluation of the prepared batches for particle size analysis, Transmission electron microscopy, entrapment efficiency, in-vitro release study etc. Further, FTIR spectroscopy, X-ray diffraction, Differential Scanning Calorimetry, Vibrational sample magnetometer (VSM) and in-vivo animal studies were also performed.

Results

VSM analysis demonstrates that the prepared nanoparticles exhibit superparamagnetic magnetic behaviour which was retained even after coating by chitosan. In-vivo studies of A-MNPs showed 19.07-fold increase in kidney uptake of AP as compared to serum post 2 h of administration in mice whereas no drug was detected in kidney and serum post 2 h administration of pure drug (free-form) indicating successful targeting to kidney as well as sustained release of AP from the formulated A-MNPs. The significant (p < 0.01) effectiveness of A-MNPs in management of hyperuricemic nephrolithiasis was observed through estimating pH and uric acid levels in urine and serum samples of mice. These findings were also confirmed by histological examination of isolated kidney samples.

Conclusion

Present investigation signifies that a simple external magnetic field is enough for targeting allopurinol to kidneys by formulating A-MNPs which further offers an effective approach for management of hyperuricemic nephrolithiasis.

Graphical Abstract

Introduction

Drug targeting to kidneys has remained a major area of research, as it performs vital functions like urine formation, maintaining homeostasis etc. and any damage to its normal functioning can cause severe problems like hypertension, inflammation, urinary tract infections, renal calculi and various others. Nephrolithiasis is one such serious disease whose prevalence has received increasing consideration of researchers in past few decades, is related to elevated serum uric acid (UA) level, increased urinary UA excretion accompanied with persistent low urine pH (below 5.5) resulting in UA calculi accumulation in renal pelvis, collecting ducts and ureters eventually causing renal pathologic alterations [1, 2]. The major risk factors for UA nephrolithiasis are low urine pH, hyperuricosuria, hyperuricemia, low urine volume, gouty diathesis, over consumption of purine rich food etc., eventually leading to excessive UA production. Allopurinol has been used for the management of hyperuricemia, which serves as a powerful xanthine oxidase (XO) inhibitor, responsible for degradation of purines to uric acid [3, 4]. Management of UA nephrolithiasis with allopurinol requires high concentration of the drug in the kidneys for longer duration, which on the other hand, can cause undesirable systemic effects and therefore allopurinol should be targeted to kidneys for obtaining maximum effect at the desired site. Nanoparticles have been extensively studied as a targeted therapeutics for various organs and in particular, magnetic nanoparticles (MNPs) have been explored for delivering therapeutics specifically to target region by application of an external magnet. Further, magnetic nanoparticles can be coated with biocompatible and biodegradable polymer that can escape the body’s defence system and shields the drug from degradation before reaching target site [5–7]. Chertok et al., found the accumulation of MNPs in gliosarcomas has been enhanced to five fold as compared to normal sites [8]. Kumar et al. selectively targeted mice kidney by placing an external magnet between the back legs and showed greater accumulation of fluorescent MNPs in kidney than nonmagnetized nanoparticles [9]. Therefore, in the present investigation, we aimed to fabricate iron oxide MNPs coated with chitosan for targeting allopurinol to kidneys by using external magnetic field for the management of hyperuricemic nephrolithiasis.

Materials

Allopurinol (AP) was generously gifted by Indoco Remedies Limited Maharashtra (India). Ferrous sulphate heptahydrate (FeSo₄.7H₂O, 99%), acetic acid glacial (99.8%) and epichlorhydrin were procured from Thomas baker (India). Ferric chloride anhydrous (FeCl₃) and chitosan were obtained from C.D.H. Pvt. Ltd., (India). Ammonium hydroxide (30%w/v) and Aciclovir were obtained from Sigma Aldrich (India). All chemicals utilized in research work were of appropriate analytical grade.

Methods

Preparation of MNPs

Iron oxide MNPs were prepared by adopting chemical co-precipitation method [10]. Weighed amount of ferrous sulphate heptahydrate (3.8 g) and anhydrous ferric chloride (4.53 g) were dissolved in distilled water, under inert atmosphere (N₂ gas) and stirred using magnetic stirrer (Tarsons SPINOT, India) at 80 °C for 1 h, followed by rapid introduction of ammonium hydroxide solution (40 ml, 30% w/v) into the mixture and stirred for another 1 h under N₂ atmosphere. After cooling down to room temperature, the precipitate was washed thrice with hot water, collected by using permanent magnet and freeze dried.

Preparation of allopurinol loaded chitosan coated magnetic nanoparticles (A-MNPs)

Prior to coating of magnetic nanoparticles (MNPs), chitosan solution (1% w/v in 0.5% acetic acid) was prepared and epichlorhydrin was added dropwise into it as a cross linker and stirred mechanically for 3 h. Afterwards allopurinol solution was added to it under vigorous mechanical stirring (Remi RQ 122, India). Further for coating, a weighed quantity of dried MNPs (500 mg) were dispersed in distilled water and the dispersion was poured into above solution and for maintaining basicity of solution, small amount of ammonium hydroxide was also added to it and stirred for 24 h. The precipitated particles were then separated by permanent magnet and freeze dried [11, 12].

Characterization of A-MNPs

Drugs content

Accurately weighed 10 mg grinded A-MNPs were added to 100 ml distilled water and the resultant suspension was shaked on mechanical shaker for 24 h, followed by filtration through 0.2 μm membrane filter. The drug loading efficiency (LE %) and entrapment efficiency (EE %) was estimated in triplicate using UV-visible Spectrophotometer at 250 nm. LE % and EE% were calculated by employing following formulas [13]:

$$\begin{array}{c}\mathrm{EE}\%=\frac{\text{conc}.\text{of allopurinol loaded in MNPs}}{\text{conc}.\text{of allopurinol intially used in formulation}}\ast 100\\ \mathrm{LE}\%=\frac{\text{concentration of allopurinol loaded in MNPs}}{\text{total}\mathrm{A}-\text{MNPs weight}}\ast 100\end{array}$$

Particle size, polydispersity index (PDI) and zeta potential

After making a dispersion of the MNPs, A-MNPs and chitosan coated but without drug MNPs (C-MNPs) was dispersed in HPLC grade water and their mean particle size, PDI and zeta potential were measured in triplicate by using Zetasizer (Malvern Instruments Limited, U.K.) based on dynamic light scattering at 25 °C and 90^o scattering angle [14, 15].

Transmission electron microscopy (TEM)

Surface morphology of A-MNPs, C-MNPs and MNPs were studied by TEM analysis. The suspension was prepared in HPLC grade water and a drop was fixed onto copper grid with carbon film coating and kept for 1 min. Excess sample was wiped off using filter paper and grid was further stained with phosphotungstic acid (2%, pH 7.0). The prepared grid was then analysed by high resolution transmission electron microscope (FEI Technai S-Twin) operated at 200 kV [16, 17].

In-vitro drug release

AP (drug) release behaviour from formulated A-MNPs was studied using USP Type 2 (paddle type, TDT 08 L, Electrolab, India) dissolution apparatus, based on dialysis technique. Lyophilized A-MNPs (equivalent to 10 mg AP, redispersed in 4 ml phosphate buffer pH 7.4) were introduced into dialysis bag (Himedia Laboratories, MWCO 12,000–14,000), previously soaked in distilled water 1 h before use. Further, dialysis bag was sealed from both ends and placed into dissolution apparatus containing phosphate buffer (pH 7.4, 500 ml, temperature 37 ± 0.5 °C, 100 r.p.m). 5 ml aliquots were collected and replenished with equal volume of fresh dissolution medium at regular intervals in order to maintain sink conditions. The absorbance of collected samples were further analysed spectrophotometrically at λmax 250 nm in triplicate for estimating amount of drug released. The drug release mechanism was evaluated by fitting the release data to different kinetic models i.e. zero order, first order, Hixson Crowell, Higuchi model (matrix) and Korsemeyer Peppas using PCP Disso v3 software [16, 18].

Fourier transform infrared (FTIR) spectral analysis

FTIR spectrums of AP (pure drug), lyophilized A-MNPs, placebo MNPs (C-MNPs) and chitosan polymer were recorded in the frequency range of 400–4000 cm⁻¹ by KBr method using FTIR spectrophotometer (IR Affinity-I, Shimadzu, Japan) for analysing the interaction between AP and carriers in the fabricated A-MNPs [19].

Differential scanning calorimetry (DSC) analysis

DSC thermograms of lyophilized A-MNPs, C-MNPs, pure allopurinol, chitosan and physical mixture of AP and chitosan polymer (P-AP-CH) in the ratio 1:1 were recorded using Differential Scanning Calorimeter (Q-10, TA Instruments, USA) for determining the physical nature of allopurinol and polymer in the formulated A-MNPs. A certain amount (2–5 mg) of each sample was poured into aluminium pan, accurately weighed, crimped and then scanned under nitrogen environment in the temperature range of 25–400 °C and heat flow rate 10 °C/min [16].

X-ray diffraction (XRD) analysis

XRD pattern of pure drug (AP), lyophilized A-MNPs, placebo MNPs (C-MNPs) were obtained on a X-ray diffractometer (Rigaku MiniFlex 2) for analyzing the physical state and interactions of drug with excipients in the fabricated A-MNPs. The samples were scanned at an ambient temperature with 2 theta range of 10° to 80° with an increasing step size and time of 0.02° and 2 s respectively using CuKα (λ-1.5405°A) monochromatic radiation at 30 kV/15 mA as X-ray source [18].

Vibrating sample magnetometer analysis

Magnetic property of MNPs and A-MNPs were analysed by Vibrating Sample Magnetometer (Lakeshore VSM 7410) at room temperature [19].

Stability studies

For carrying out the stability studies, the prepared A-MNPs was stored at different conditions i.e. 4° ± 2 °C, 25° ± 2 °C and 45° ± 2 °C for 3 months and the samples were analysed at the end of 1, 2 and 3 month for drug content and particle size [20].

In vivo animal studies

For performing in vivo animal studies, approval was acquired from Institutional Animal Ethics Committee (IAEC), G.J.U S&T, Hisar, India (Registration No: 0436) and the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) of Ministry of Environment and Forests (Animal Welfare Division), Government of India were strictly followed. Swiss albino mice (25–30 g) were acquired from Disease Free Small Animal House, LUVAS, Hisar, India. Prior to the commencement of study, animals were acclimatized (1 week period) to the laboratory environment.

Potassium oxonate (uricase inhibitor) was employed for inducing hyperuricemia in mice which not only increases the risk of UA nephropathy but also responsible for causing nephrolithiasis [21, 22]. For induction, an intraperitoneal injection (i.p) of potassium oxonate (250 mg/kg) dissolved in 0.9% NaCl solution was given to each animal for 7 successive days.

Study design: Animals were categorized in 5 groups having six animals in each group (n = 6).

• Group 1: Control group, normal saline was given to animals for 7 consecutive days.

• Group 2: Inducer, Potassium oxonate (250 mg/kg, PO) was administered intraperitoneally for 7 consecutive days.

• Group 3: Pure drug, allopurinol (5 mg/kg) was administered orally for 7 consecutive days after 1 h of PO administration (250 mg/kg, i.p).

• Group 4: Formulation, A-MNPs was administered by intravenous injection (i.v) for 7 consecutive days after 1 h of PO administration (250 mg/kg, i.p). A circular magnet was placed between the legs of mice for targeting kidney [9]. The dose of A-MNPs (equivalent to 4 mg/kg, AP) was based on the bioavailability of AP by oral route.

• Group 5: Placebo, C-MNPswas administered for (i.v) 7 consecutive days after 1 h of PO administration (250 mg/kg, i.p).

UA supersaturation in urine correlates with the risk for the development of UA stones. Further, UA supersaturation in urine depends upon increased UA production in the body as well as reduced urine pH [23, 24]. Both these factor are considered as risk factor for UA stone formation. Therefore, we have assessed UA levels in urine and serum samples as well as the pH of urine samples. Moreover, histological studies of kidney obtained after sacrifice of animals were also conducted for evaluating nephropathy as well as nephrolithiasis.

Determination of uric acid level

After treatment of 7 days, food and water were withdrawn from the animal cages. Blood samples were collected and kept aside for clotting for 1 h and centrifuged (9000 rpm, 15 min) for serum collection. The serum and urine samples were stored at −20 °C until further processing. The levels of uric acid (UA) were quantified in serum and urine samples at 520 nm by employing enzymatic colorimetric method [25]. Thereafter, mice were sacrificed using cervical decapitation under the influence of deep anesthesia and immediately their kidneys were harvested, cleaned with saline to get rid of excess surface blood and stored at −20 °C until further analysis.

Determination of urine pH

The pH of collected urine samples were determined (URiSCAN optima).

Histological study

After isolating kidney from each animal, it was immediately preserved in formalin solution (10%) and later fixed into paraffin. Kidney sections were cut and stained using hematoxylin and eosin (H-E) and analysed under microscope for histopathological examination.

Kidney uptake potential

HPLC analysis was performed for estimating amount of drug in kidneys as well as in serum samples (n = 3). Before carrying HPLC analysis, serum and kidney samples were prepared. Serum samples (100 μl) were mixed with aciclovir solution (internal standard, 10 μl), followed by addition of 10% trichloroacetic acid solution (100 μl) and shaken well and finally centrifuged (9000 rpm, 15 min) for separating supernatant [26]. On the other hand, samples of kidneys were then weighed, homogenised using tissue homogenizer in dimethyl sulfoxide (5 volumes) and later centrifuged (9000 rpm, 15 min) for separating supernatant [27, 28]. Further the supernatant obtained from serum and kidney samples were filtered (0.22 μm, syringe filter) and injected into HPLC (30 μl each) for estimating drug content. The HPLC system was consisted of ZORBAX column (SB-C18, 5 μm, 4.6 × 150 mm) and the mobile phase comprised of 0.02 M sodium acetate solution (pH 4.5 adjusted with 30% acetic acid) at a constant flow rate of 1 ml/min. Only freshly prepared mobile phase (degassed and filtered) was utilized throughout the study. The UV wavelength was kept at 254 nm and total run time for sample was 5 min [29].

Statistical analysis

The statistical analysis of in vivo data was conducted via utilizing a GraphPad InStat software. The data obtained was presented as mean ± standard error of mean and one way analysis (ANOVA) with dunnett test was also performed for determining the difference among values. All groups were compared with PO group (inducer) and p values less than 0.05 was taken as statistical significant.

Results

Entrapment efficiency

The entrapment and loading efficiency of A-MNPs were found to be 57.55 ± 0.05% and 27.35 ± 0.02% (mean ± S.D) respectively.

Particle size, polydispersity index (PDI) and zeta potential

Particle size, PDI and zeta potential observed by zetasizer for MNPs, C-MNPs and A-MNPs are displayed in Table 1. All fabricated MNPs were found to be in nano range (less than 250 nm) and PDI values far from 1 indicate dispersion to be in homogenous state. Zeta potential is a measure of surface charge and higher values of ZP of fabricated MNPs indicates existence of large repulsive forces between particles in dispersion which favours less aggregation and higher stability [30]. Moreover, increase in particle size of C-MNPs and A-MNPs denote successful coating of polymer and drug [31].

Table 1.

Particle size, PDI and Zeta potential of MNPs (n = 3)

S.no	Formulation	Particle size (nm) ± SD	PDI ± SD	Zeta potential (mV) ± SD
1	MNPs	194.7 ± 6.18	0.473 ± 0.106	−20.9 ± 0.25
2	C-MNPs	212.6 ± 9.92	0.526 ± 0.017	−19.4 ± 0.20
3	A-MNPs	209.8 ± 5.54	0.504 ± 0.012	−23.6 ± 0.32

Transmission electron microscopy (TEM)

TEM micrographs of MNPs, C-MNPs and A-MNPs have been presented in Fig. 1 which revealed that particles were uniform and almost spherical in shape. Particle size of prepared MNPs was found in the range of 38.21–53.40 nm while particle size of C-MNPs and A-MNPs was found in the range of 44.40–86.41 nm and 46.40–91.65 nm respectively, which was significantly more than MNPs, indicating successful coating of chitosan and drug incorporation. The particle size obtained from Dynamic light scattering (DLS) technique (zetasizer) was quite larger than by TEM imaging which is possibly because of dehydration of MNPs during sample preparation for TEM analysis. On the other hand, DLS technique records the particle size as hydrodynamic diameter which generally comes to be higher than measured TEM analysis [32].

Fig. 1.

TEM micrograph of a MNPs b C-MNPs and c A-MNPs

In vitro drug release study

In vitro release profile of allopurinol from formulated A-MNPs is shown in Fig. 2. 80.54 ± 2.29% of drug was released in a time span of 12 h. Further the data procured from release study was fitted into different release kinetic equations which gave correlation coefficients values (R²) of 0.9814, 0.9844, 0.9856, 0.9833 and 0.9529 for Hixson–Crowell, Korsemeyer–Peppas, Higuchi, first order and zero order models, respectively. The higher R² values for Higuchi model suggests drug release via A-MNPs was dominated by diffusion through swelled polymer matrix and the value of release component was found to be 0.67 which signifies non-Fickian drug transport [33]. Chitosan nanoparticles prepared by Chavan et al. also showed the same drug release pattern following Higuchi kinetics [34].

Fig. 2.

In vitro release profile of allopurinol drug with curve fitting of data

Fourier transform infrared (FTIR) spectral analysis

Figure 3 represents the FTIR spectrum of pure AP, chitosan, MNPs, A-MNPs and C-MNPs. FTIR spectra of AP showed characteristic peaks at 3360 and 3043 cm⁻¹ corresponding to NH stretching band and CH stretching vibrations, respectively. Peaks 1705 cm⁻¹ could be attributed to CO stretching vibration, peak at 1593 cm⁻¹ relates to ring vibration and peaks at 1240, 1160, 915, 815 and 783 cm⁻¹ relates to CH in plane deformation. The FTIR spectra of chitosan depicted a broad peak in the range of 3400–3300 cm⁻¹, ascribed to OH group and peaks at 2946, 1646 and 1371 cm⁻¹ corresponding to CH stretching, NH bending and CO stretching of 1^o alcoholic group of chitosan, respectively [18]. IR spectrum of MNPs showed a characteristic peak of Fe-O at 576 cm⁻¹ and the characteristic peaks of chitosan and MNPs were present in C-MNPs, thus it can be inferred that MNPs were effectively coated with polymer. Further, spectra of A-MNPs showed existence of all the characteristic peaks of chitosan, MNPs and allopurinol which confirms successful loading of drug in magnetic nanoparticles as well as polymer coating.

Fig. 3.

FTIR spectra of allopurinol (AP), chitosan, A-MNPs, C-MNPs and MNPs

Differential scanning calorimetry (DSC) analysis

Figure 4 depicts the DSC thermograms of AP, chitosan, P-AP-CH, C-MNPs and A-MNPs. DSC curve of allopurinol revealed a sharp endothermic peak at 383.3 °C, indicating its crystalline state and melting point. The DSC curve of chitosan depicts a broad endothermic peak at 102.25 °C followed by its degradation represented by an exothermic peak at 300.47 °C. DSC thermogram of P-AP-CH showed the respective peaks of chitosan and AP at their respective places which indicates no interaction among AP and polymer. In case, of C-MNPs, again the peaks of chitosan were present at their respective position indicating the coating of chitosan on MNPs. A -MNPs thermogram shows a slight shift in the endothermic peak of chitosan to 99.95 °C that may have occurred due to formulation process. Also, drug’s crystalline endothermic peak was converted into a broad peak indicating the change of crystalline state of AP in the formulation, signifying entrapment of drug in MNPs [16, 18].

Fig. 4.

DSC curves of allopurinol (AP), physical mixture of AP and chitosan in ratio 1:1 (P-AP-CH), A-MNPs and C-MNPs

X-ray diffraction (XRD) analysis

The X-ray diffractogram of allopurinol as shown in Fig. 5, reveals sharp characteristic peaks at 2 theta values of 10.53^o, 11.93^o, 14.75^o, 17.25^o, 20.06^o, 25.68^o, 27.87^o and 34.90^o, indicating its crystalline nature. Amorphous nature of chitosan was shown in XRD pattern of C-MNPs. The characteristic peaks of AP were not present in X-ray diffractogram of A-MNPs which again demonstrates reduction in crystallinity during incorporation of drug into the MNPs [16, 18].

Fig. 5.

XRD patterns of allopurinol, A-MNPs and C-MNPs

Vibrating sample magnetometer

Figure 6 displays the magnetization curve of MNPs and A-MNPs. Magnetic curve of MNPs and A-MNPs showed coercivity and retentivity closer to zero at room temperature, which indicates that the prepared nanoparticles have superparamagnetic property and the saturation magnetization of A-MNPs was reduced as compare to MNPs, which may have occurred due to polymer coating of chitosan and drug loading over naked MNPs [35, 36].

Fig. 6.

VSM graphs of magnetic nanoparticles (MNPs) and allopurinol loaded chitosan coated magnetic nanoparticles (A-MNPs)

Stability studies

A-MNPs samples didn’t show any significant changes in drug content and particle size even after storing at 4° ± 2 °C, 25° ± 2 °C and 45° ± 2 °C for 3 months.

In vivo animal studies

Determination of uric acid level and urine pH level

Uric acid level was estimated in serum and urine samples of mice (n = 6) after 7 days by employing uricase method. In control group, serum uric acid (SUA) concentration was found to be 1.15 ± 0.05 mg/dL, whereas SUA concentration was significantly (p < 0.01) increased to 7.64 ± 0.33 mg/dL in inducer group as PO inhibits the enzyme uricase responsible for breakdown of UA, indicating successful establishment of hyperuricemia [21, 22]. As compared to inducer group, SUA level in formulation (A-MNPs) treated group was reduced significantly (p < 0.01) in hyperuricemic mice to 1.66 ± 0.13 mg/dL, due to the AP in A-MNPs which inhibits XO enzyme responsible for UA formation, signifying effectiveness of formulated A-MNPs (Fig. 7a).

Fig. 7.

a Effect on mice serum uric acid level. Values are depicted as mean ± SEM (n = 6). ‘*’ p < 0.01 inducer vs control group. ‘a’ p < 0.01 vs inducer group. ‘b’ p > 0.05 vs inducer group. ‘#’ p < 0.05 AP vs A-MNPs. b Effect on mice urine uric acid level. Values are depicted as mean ± SEM (n = 6). ‘*’ p < 0.01 inducer vs control group. ‘a’ p < 0.01 vs inducer group. ‘b’ p > 0.05 vs inducer group. ‘##’ p < 0.01 AP vs A-MNPs. c Effect on mice pH of urine samples. Values are depicted as mean ± SEM (n = 6). ‘*’ p < 0.01 inducer vs control group. ‘a’ p < 0.01 vs inducer group. ‘b’ p > 0.05 vs inducer group. ‘##’ p < 0.01 AP vs A-MNPs

Thereafter uric acid level was measured in mice urine and urine uric acid (UUA) concentration for control and inducer animal group were found to be 0.39 ± 0.10 mg/dL and 5.39 ± 0.12 mg/dL respectively. A significant difference (p < 0.01) between UUA levels of control and inducer group depicts development of hyperuricosuria successfully. The UUA level was decreased significantly (p < 0.01) in A-MNPs treated group to 2.08 ± 0.13 mg/dL showing effectiveness of A-MNPs in treating hyperuricemic nephropathy (Fig. 7b).

pH level of collected urine samples of mice were determined and found to be 8.2 ± 0.10 for control group. The pH level in inducer group was significantly (p < 0.01) reduced to 5.1 ± 0.14 which again confirms the model establishment. The pH level of A-MNPs treated group was found to be 7.6 ± 0.11 (p < 0.01) which represents efficacy of formulated A-MNPs (Fig. 7c).

However, the SUA and UUA levels in pure drug group was observed to be higher (p < 0.05 and p < 0.01, AP vs A-MNPs) and pH levels (p < 0.01, AP vs A-MNPs) were found to be lesser than A-MNPs group further confirming more effectiveness of A-MNPs as compared to pure drug at the same bioavailable dose. SUA, UUA and pH levels of placebo group showed insignificant difference (p > 0.05) from inducer group, depicting that excipients had no impact on treatment process.

Histological study

Histological analysis of kidney further confirms the above findings. The H-E stained kidney section of control group (Fig. 8) showed normal renal parenchyma with maintained cortical and medullary architecture having unremarkable glomeruli and interstitial tissue along with no evidence of inflammation, tubulitis and necrosis. By contrast, in potassium oxonate (inducer) treated group, kidney damage was characterized by ruptured glomeruli interstitial edema as well as infiltration of lymphocytes and plasma cell seen as distinct patches. Also, evidence of tubulitis and tubular casts depicts the establishment of hyperuricemic nephropathy in mice. C-MNPs treated group showed similar kidney damage like PO group and didn’t showed any healing, thus indicating no influence of excipients on the treatment process. Histological examination of AP treated group revealed only shrinkage of the glomerular tufts along with lesser interstitial lymphoplasmocytic infiltrate, lesser tubulitis and interstitial edema, indicating the effectiveness of AP. Again effectiveness of our developed A-MNPs formulation is indicated by intact glomeruli as well as minimal interstitial edema and lymphoplasmacytic infiltrate.

Fig. 8.

Histological images of mice kidney isolated from control, PO, C-MNPs, AP and A-MNPs group

Kidney uptake potential

After 2 h of A-MNPs administration, the concentration of AP in kidney was found to be 7.44 μg/g and in serum was 0.39 μg/ml, which reveals a 19.07 fold increase in amount of AP targeted to kidney as compared to AP in serum. Moreover, no drug was detected post 2 h administration of pure allopurinol in kidney as well as in serum which further confirms successful sustained release of the drug in case of A-MNPs apart from targeting.

Conclusion

The present work demonstrates development of allopurinol loaded chitosan coated magnetic nanoparticles (A-MNPs) as an effective approach for targeting kidney and management of hyperuricemic nephrolithiasis in preclinical studies. The developed MNPs were coated with hydrophilic chitosan polymer for protecting MNPs from earlier removal by body’s defense system. In vivo studies as well as histological examination of isolated kidneys showed 19.07 fold increased availability of allopurinol drug in kidneys as compared to serum and significant drug release for an extended period of time. The present formulation of A-MNPs can be further studied in clinical setting for successful kidney targeting of the allopurinol for the effective therapeutic management of hyperuricemic nephrolithiasis.

Abbreviations

A-MNPs

Allopurinol loaded chitosan coated magnetic nanoparticles

AP

Allopurinol

C-MNPs

Chitosan coated magnetic nanoparticles (without drug)

DSC

Differential Scanning Calorimetry

FTIR

Fourier Transform Infrared

HPLC

High Performance Liquid Chromatography

MNPs

Magnetic nanoparticles (without drug and polymer coating)

MWCO

Molecular weight cut off

P-AP-CH

Physical mixture of AP and chitosan polymer

PO

Potassium oxonate

SUA

Serum uric acid

TEM

Transmission Electron Microscopy

UA

Uric acid

UUA

Urine uric acid

VSM

Vibrating sample magnetometer

XO

Xanthine oxidase

XRD

X-Ray Diffraction

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interest.