1. INTRODUCTION:

A planetary well-being problem is Chronic-Kidney-Disease (CKD) that leads to a sharp financial stack for nations and patients, and a lethal phenomenon as well. Majority of the diseased persons with CKD are approximately inclined to cardiovascular diseases, early death and their quality of life is severely limited attributable for treatment management such as dialysis.^1,2,3

World-wide deaths in 2017 due to CKD was accounted for and it is found to be 1.2 million, and the passing rate expanded from 2007 to 2017 by 33.7% entirely⁴. Death number of an over the top 7000 - 10 000 from end-stage kidney infection (ESKD) amid the starting months of the COVID-19 widespread happened, as per the information based on ESKD mortality rate inclination from 2016 - 2019. People are at an enhanced risk of death from COVID-19 who are suffering from ESKD. COVID-19 related results are worse mostly for aged people due to the multiple treatment-resistant conditions, Reduced immune system are also an outcome for a few. About 800 000 patients are reported with ESKD has been reported and this information is analyzed from national registry by Researcher of Centers for Medicare and Medicaid Administrations from Feb-1-2020, Additionally 8.7 to 12.9 deaths per 1000 patients with ESKD analyzed through the august and this have been became expectable phenomenon based on the previous year’s data⁵. Critical problems in humans and animals arise due to incapable kidney function which results in the aggregation of toxins in the body which are uremic in nature. Presently, in spite of procuring medications such as dialysis for CKD patients, the viability is constrained and shows low people compliance. Understanding who is habitually dialyzed for the urea clearance is lower by six times when analyzing its strength with a well-being person. Thus, urinary toxins aggregate into the body due to inadequate kidney function.

An amino acid which is constituted in the food named - Tyrosine, regenerates by gut microbiota into p-cresol and it leads to the formation of uremic toxins, if it cannot be able to excrete from the body¹³. p-cresol sulfate has been coupled to harm the tubular cells mediated by oxidative stress in kidney cells⁶, fibrosis happen due to the actuation of epithelial-to-mesenchymal move⁷, overall renal damage with inflammation also associated⁸, an initiate blood vessel calcification was appeared by a 7-week presentation to p-cresol sulfate or indoxyl sulfate in a CKD rodent demonstrate, impaired glucose homeostasis and a decrease in GLUT1 expression is a prodiabetic condition also activates by this exposure⁹, insulin resistance and redistribution of lipids in the muscle and hepatic tissues also activates by the administration of p-cresol sulfate to healthy mice¹⁰, ESRD patients had less bacteria able of modifying dietary fiber to short-chain fatty acids and bacteria expressing hydroxyphenylacetate decarboxylase, which is an enzyme responsible for the amino acid - tyrosine conversion to p-cresol¹¹, Levels of p-cresol sulfate in cerebrospinal fluid (CSF) were higher than circulating levels and this observation revealed by a study of 2020 conducted on patients of Parkinson’s disease (PD).¹²

In 1991, in order to adsorption of uremic toxins AST-120 was formulated which is an oral spherical carbonaceous adsorbent. AST-120 smothers the arrangement of indoxyl sulfate – another uremic poison within the body, but not p-cresol from the digestive tract appeared by an animal study¹³. For CKD patients - in order to discover a way better to adsorb and remove the p-cresol from the gastrointestinal (GI) fluid is therefore of great interest by studying the new molecules and formulations for adsorption.

Nanosponge (NS), with nano-sized pores, is one of the progressed nanomaterials in cutting edge revelations of nanotechnology by changing the synthesis and control of nano-sized materials. Though, adsorption of toxic molecules, carrying of biocatalysts, enzyme and gases immobilization and drug delivery are quite wider potential uses of NS^14,15. Hyper-cross-linked polystyrene-based, carbon-coated metallic-based, silicone-based, titanium-based and cyclodextrin (CD) based are widely studied in 5-main categories of NS¹⁴. Among all the Nanosponges. The most beneficial system is found to be CD-based. Because CD-based systems for many years have been used as pharmaceutical excipients and are natural biocompatible polymers^16,17,18. CDs can transport tiny molecules by trapping them in their inner cavities thanks to their special molecular structure. In order to enhance drug solubility, mask smell and taste, reduce systemic and local side effects, improve stability and absorption, control drug release profile and enhance permeability of medicate through organic boundaries with by and large recognized as secure (GRAS) status emphasizing security in human utilize^{17,19,20,21,22}. CDs can encapsulate harmful particles in their inside cavities and are considered as exceedingly permeable CD-NS definitions can be shining nanomaterials, other than their utilize as medicate conveyance framework components and CD-NS may moreover be promising nanomaterial definitions for adsorption of the harmful atoms, Besides the use as drug delivery system components.

CD-NS, covalently connected with cross linkers in the formulation which are a kind of new systems with multiple units not only single CD. Over the past decade CD-NS techniques have heightened, particularly after a European Commission report spotlighted that it is a promising system in delivery of drugs^23,24. Research on different groups have worked on CD-NS as anticancer drug’s carrier which include paclitaxel²⁵, camptothecin²⁷, tamoxifen²⁶, antivirals (acyclovir)²⁸, antifungals (itraconazole)³⁰, curcumin²⁹, anti-inflammatory (resveratrol)³¹, hormones (melatonin)³³ and gases(oxygen)³². In spite of the virulent factors from the body³⁵ and studies on clearance of water from the organic contaminants,³⁴available in the literature by using NS, within the field of poisonous particles clearance, the utilization of CD-NS is exceptionally restricted. CD-NS can expel harmful atoms from GI liquid is a theory based on the over study.

Different drug delivery systems appear to be a dynamic region of research and advancement by consolidation of natural polymers due to self-evident preferences counting being reasonable, ready availability⁵⁰, eco-friendly, able of chemical adjustments, potentially degradable and biocompatible due to their natural root.⁵³ Natural polymers which have been investigated for their promising potential in Gastric-specific drug delivery incorporate alginates, chitosan, xanthan gums, starch etc.⁵⁰. From brown algae, Alginate is naturally occurring polysaccharide polymer gotten which comprises of two diverse units, β-Mannuronic acid (M units) and α-L glucuronic acid (G units) residues. It is additionally known as complex polysaccharide whose composition shifts on the basis of the proportions of its monomeric units⁵¹.

Sodium alginate (SA) is a family of linear unbranched natural polysaccharides, the sodium salt of alginic acid⁴⁹ that's exceptionally promising and has been widely exploited within the pharmaceutical industry, since of its tailor‐made to suit the requests of applications⁵². It is wealthy within the carboxyl group and is effectively bound with a positively charged drug. It is a low harmful, biocompatible and inexpensive biopolymer, which empowers it to be broadly utilized in medicine⁵⁵. The two monomers of sodium alginate contain a free carboxylic group(-COO-). During the hyper-cross-linking process with β-cyclodextrin, there's an interaction between the particles of β-cyclodextrin and the free carboxylic group(-COO- ) of sodium alginate within the G block of the alginate chain. As a result, it produces a rigid structure that shapes the nanoparticles⁵⁴.

In-vitro assessment of the biosafety, viability and biocompatibility of four CD-NS formulations by using different proportions of Sodium alginate as cross linker in adsorbing p-cresol which may demonstrate a poisonous particle from GI fluid in CKD patients which is the most significant objective of this research. Optimum, CD-NS was chosen by assessing it’s - GI physical steadiness, adsorption efficiency of phenol (p-cresol), physico-chemical characteristics and absence of systemic uptake and safety as well through in-vitro studies.

2. MATERIALS AND METHODS:

2.1 MATERIALS:

From R.P Chemicals (Thane): β-CD were obtained, Other substances such as : Sodium alginate, buffer salts, all chemicals and solvents were provided by premier pvt, ltd, Hyderabad. Distilled water was obtained from Deccan School of Pharmacy, Hyderabad. Pepsin, Pancreatin and α-amylase enzymes was a kind gift from Enzymes Bioscience pvt, ltd, Gujarat.

2.2 METHODS:

2.2.1 Preformulation Studies:

Melting Point :

Assurance of dissolving point of sedate was done by capillary tube utilizing dissolving point device. At first the test was carefully organized into the capillary tube to stack. To the foot of this capillary tube the powder was pushed, and then sample tubes are kept in to one of the sample positions lots located on the top of the instrument, the heating stand was preheated to specified temperature just few degrees below the expected melting point of the sample and observe the changes in the sample until it start melting and compare it with reference sample.

2.2.2 Formulation development:

Primarily, In DMSO β-CD and sodium alginate is dissolved in different ratios such as 1:1, 1:2, 1:4 and 1:8 and named each definition as NS1, NS2, NS3 and NS4 separately at room temperature.

Acetone is used to eliminate the unreacted reagents and solvents that are used during the preparation such as DMSO from the preparation. For 24 h the definitions were washed in Soxhlet with acetone and were dried in a hot air oven overnight at 80 °C to dispose of the acetone and finally put away in a dried container.

Table 1 : Composition Of Nanosponges Formulation

S. No	Ingredients	NS1 (1:1)	NS2 (1:2)	NS3 (1:4)	NS4 (1:8)
1.	β-Cyclodextrin (gms)	75	50	50	50
2.	Sodium Alginate (gms)	75	100	200	400
3.	Dimethyl sulfoxide (ml)	300	200	200	200

2.2.3 Evaluation of Formulation

2.2.3.1 Evaluation of Nanosponges :

FTIR – Studies for Complex formation :

FTIR thinks about were performed on Beta-Cyclodextrin, Sodium alginate and of the upgraded detailing. With KBr powder around 0.1 to 1.0 % of the test is well blended and after that pulverized finely and kept into a pellet shaping pass on. A drive of roughly 8-tons is connected beneath a vacuum of a few mm Hg for many minutes from clear pellets. To arrange dampness degassing is performed from the KBr powder. The foundation can be measured with a purge pellet holder embedded into the test chamber when performing estimations. Be that as it may, establishment estimations on a pellet holder of instrument with a pellet of KBr as because it contains no test, can change for infrared light diffusing misfortunes within the pellet and for dampness adsorbed on the KBr. The tests were analyzed between wave number 4000 and 400 cm-1

Swelling Ability :

In terms of the maximum level of hydration the swelling ability of NS was examined by weighing the amount of absorbed water by NSs.³⁶

Solubility :

NS properties of solvency assessed in several solvents. 1 mg of NS was included in 5 mL of dissolvable and overnight sonicated for this reason. At that point in this solubility detect by the dissolvability of NS and was inspected outwardly.³⁶

2.2.3.2 NS Dispersion Characterization after Preparation :

As these arranged NS definitions arranged to manage orally, Fluid scattering of NS were characterized by assessing mean molecule measure (nm), surface charge (mV), polydispersity index (PDI) and physical characterization of steadiness of NSs in reenacted GI liquids. Also, an image of each aqueous NS-dispersion preparation was carried out by using Scanning electron microscopy.

Aqueous NS dispersion Preparation :

In distilled water NS was dispersed in (1 mg/mL) and set under magnetic stirring at 550 rpm for 4 hours. Finally, the conceivable and non-dispersible polymer contents were collected and expelled out by utilizing film filtration with pore estimate 0.45 μm.³⁶

Mean Particle Size estimation and PDI estimation:

Mean particle size(MPS) and PDI were examined by using Nano Particle Analyzer. For this purpose, 0.5 mL NS dispersion was diluted by adding 0.5 mL distilled water and was put in the folded capillary zeta cell. Estimation was realized by NANO PARTICA - Instrument of Nano Particle Analyzer : SZ - 100 by HORIBA Scientific at room temperature utilization(n = 3).

Charge of Surface estimation :

Charge of surface estimation of NS was done by utilizing NANO PARTICA - Instrument of Nano Molecule Analyzer : SZ - 100 by HORIBA as zeta potential(ZP). This zeta potential was measured in mV units. Utilization of Capillary Zeta Cells containing NS scattering were used for the surface charge estimation as well by the test which was arranged for mean molecule estimate and PDI estimation as well, and zeta potentials of each test were measured taking after the estimated estimation (n = 3).

Imaging of NS:

Scanning Electron Microscopy lens (SEM) was utilized to imaged NS for analyzing the surface morphology and affirm already measured molecule estimate by the Nano Molecule Analyzer instrument. For this preparation, Scattered NS were coated with gold at a thickness of 100 Å. As this Coating handle diminishes the surface charge of NSs and this leads to the coming about in clearer pictures. S-3700N HITACHI - High Technologies : Utilization of SEM instruments takes place for imaging of NS.

2.2.3.3 Preparation of Simulated Biological Fluids:

1. Simulated Gastric Fluid:

● Weigh 2g of NaCl and 3.2g of Pepsin inferred from stomach mucosa 800 to 1200 units per milligram

● Add 7ml concentrated HCl to dissolve

● Transfer into 1000ml volumetric flask Add deionized/distilled water into 1000ml volumetric carafe in drops and Make up to the check with deionized/distilled water.

● Test the pH with a pH meter and the pH ought to be a pH of 1.199.³⁷

2. Simulated Intestinal Fluid:

● Weigh 6.8g of monobasic potassium phosphate into a 500ml beaker

● Add 250ml of refined water and blend to dissolve.

● Add 77ml of 0.2N NaOH. Transfer the arrangement into a 1000ml volumetric flask

● Add 300ml of water Add 1g of Pancreatin to the solution.

● Measure the pH (The pH will be in locale of 4.5-4.6 at 25oC) Correct the pH of the arrangement to pH 6 by including 0.2N NaOH to extend the pH esteem to 6.0 (0.2N HCl can too be utilized in the event that the arrangement is basic).

● Make up the arrangement to the 1000ml mark.³⁸

3. Simulated Salivary Fluid (SSF):

● The composition for Artificially prepared salivary liquid was 3.775 mL of KCl, 0.925 mL of KH₂PO₄, 1.7 mL of NaHCO₃, 0.125 mL of MgCl₂ · (H₂O)₆ and 0.015 mL of (NH₄)₂CO₃. F

● Simulated salivary medium, the medium was arranged by blending 14 mL of Artificially prepared salivary liquid,

● 0.25 mL of an α-amylase arrangement (1.3 mg/mL),

● 0.10 mL of 0.3M CaCl₂ and 5.65 mL of distilled water.

● And at that point the pH of the blend was balanced to 6.8±0.2 by expansion of 0.1 mol/L HCl solution.³⁹

2.2.3.4 Evaluation of Simulated Biological Fluids:

A. Evaluation of Simulated Gastric and Intestinal Fluids:

1. Determination of pH⁴⁷:

The pH of the artificial biological fluids was determined using a pH meter (pH Meter Model EQ 610). Calibration of the pH meter has been done by using buffer solutions. The pH obtained were the average of three determinations.

2. Measurements of the osmotic pressure (osmolality):

The osmotic weight estimation of artificial gastric and intestinal liquid was performed agreeing to the 4th European Pharmacopoeia (Ph. Eur. 4) monograph, in which it specifies the freezing point method. The osmotic pressure of each dissolution medium was determined in triplicate.⁴¹

3. a. Buffer capacity of Simulated Intestinal fluid:

The buffer capacity was determined according to the titration method and reported as the volume of strong acid added to one liter buffer solution to lower its pH by one unit. This was determined by adding 1 N hydrochloric acid solution dropwise from a 25 mL buret into the 100 mL sample buffer solution, with constant stirring, until the pH decreased by one pH unit. The examinations were performed in triplicate for each biological fluid.⁴¹

b. Buffer capacity of Simulated Gastric Fluid:

By carrying out titration with NaOH, the buffer capacity of tests were determined. The substance of the gastric liquid is more-safe to diminish in pH. It ought to be noted when a strong acid is added than to an increase in pH when an equivalent molar amount of a strong base is added^43,44.

B. Evaluation Of Simulated Salivary Fluid:

1. Determination of pH :

2. Evaluation of Relative Density :

The relative densities of each formulation were decided employing a pycnometer. The weight of the purge pycnometer was measured. Filled into the pycnometer and then each sample was weighed. Interpretations were taken at room temperature. Each measurement was then run in triplicates. Apparent density was calculated by the following equation:

ρ = W₂-W₁/C

where,

ρ = apparent density, W₂ = weight of filled pycnometer (gram), W₁ = weight of empty dry pycnometer (gram), C = Capacity of the pycnometer in millilitre at 20^oC (ml³).

3. Evaluation of Viscosity :

The consistency of each formulation was decided by employing a glass U-tube viscometer, measure G. Each test was filled through tube L to marginally over the check “G”. The tube was set vertically and the fluid was at that point sucked to a point around 5 mm over the stamp E. After discharging weight and suction, the time taken for the foot of the meniscus to drop from the top of check E to the point of check F was measured. The interpretations were taken at room temperature. Viscosity was evaluated by the equation:

η_w/η_c = (ρ_w×t_w)/(ρ_c×t_c)

where

η_w = viscosity of water (centipoises), η_c = viscosity of test (centipoises), ρ_w = density of water (g/ml3), ρ_c = density of test (g/ml3), t_w = time taken for the bottom of the meniscus to drop from the top edge of stamp E to the top edge of stamp F (diminutive) for water, t_c = time taken for the foot of the meniscus to drop from the top edge of stamp E to the top edge of check F (minute) for test.⁴⁰

2.2.4 NS Stability examination in artificially prepared biological fluids in-vitro:

In Simulated gastric fluid (SGF) (pH 1.2) preparation and in Simulated intestinal fluid (SIF) (pH 6.8) preparation, The NS were dispersed in it and mean molecule estimate, PDI and surface charge changes were explored to decide the physical stability of NSs in GI after orally administering of the formulation.

NS was scattered as (1 mg/mL) in artificially prepared biological fluids in-vitro i,e.(SGF and SIF) under a magnetic blending at 550 rpm for 4 h. At certain time centers the tests from each NS scattering were taken as (Fresh, 3rd, 5th, 10th and 20th days), and the mean particle estimate, PDI and charge of surface parameters were measured at each time interval. Lastly, Changes in mean particle size, PDI and surface charge of NS were estimated.

These Nanosponges formulations are planned to be administered orally because the Oral route is considered most natural, uncomplicated, convenient and safe due to its patient compliance⁴⁶, pain avoidance, ease of ingestion and versatility⁴⁸. For this purpose the formulation is incubated in Simulated salivary fluid (SSF) with amylase, as well reviewed to evaluate the effect of amylase on NSs stability.

2.2.5 Adsorption efficiency estimation of phenol in Phenol Solutions of Different Concentrations:

Preparation Of Different Phenol Solution Concentrations:

In vitro analysis has been done by measuring the Phenol adsorption capacity of NS in 2 different media i,e., SGF and SIF. First phenol solutions of different concentrations for this purpose were prepared as (5 ppm, 10 ppm, 20 ppm and 40 ppm) in SGF and SIF media as follows in below table.

Determination Of the Adsorption Extent By HPLC – Analysis:

In 4 mL of solution of phenol, 80 mg of Nanosponges were dispersed and kept on a magnetic stirrer at 550 rpm for about 4 h at room temperature. By the end of 4 h, NS dispersions were centrifuged at 3,500 rpm for 15 min and the separated upper layer that is supernatant was separated in order to remove NS from the media.³⁶By the method High-performance liquid chromatography (HPLC), The free phenol for each NS dispersions was measured in the supernatant and adsorbed phenol (%) was calculated indirectly by subtracting the measured phenol amount from the initial phenol quantity.³⁶

Quantification by HPLC consisted of Agilent 1100 HPLC system with a reverse phase C18 column (250 mmx4.6 mm, 5 µm particle size), a mobile phase of water: acetonitrile (50:50 v/v) and detected with Ultraviolet (UV) detector 280 nm wavelength.⁴²

3. RESULTS AND DISCUSSION:

3.1 Preformulation Studies :

Melting Point Determination:

Table 3 : Melting point

Reported Melting point	Observed Melting point
290-300^oC	292^OC

The melting point was found to be in the range of Beta-Cyclodextrin is 290-293^oC. The measured melting point was found to be 292^oC. This indicates the purity of the sample. Any impurity if present will cause variation in the melting point of the given sample.

3.2 Formulation Development:

Fig-1 : NS1 (1:1)

Fig-2 : NS2 (1:2)

Fig-3 : NS3 (1:4)

Fig-4 : NS4 (1:8)

In order to dispose of the acetone the formulations were dried in a hot air oven overnight at 80°C and at long last the substances were put away in a dried container.

Fig-5 : Nanosponges Formulation Of Different Ratios

Sodium alginate (SA)-based β-cyclodextrin (β-CD) Nanosponges were prepared and after that stored in dried bottles.

3.3 Nanosponges Evaluation:

3.3.1 Complex Formation estimation from FTIR:

Fig- 6 : FTIR of β-Cyclodextrin:

Fig-7 : FTIR of Sodium Alginate

Fig-8 : FTIR of β-Cyclodextrin Based Nanosponge

The beta-cyclodextrin and its combination with sodium alginate formulation were subjected to FTIR. It was observed that :

● The number of OH-groups of β-CD get reduced from 6-OH groups to 2-OH groups which can be concluded by observing at the peak range between 3500-3250 cm-1 and

● The peak height decreased at the peak range between 1750-1500 cm-1, which indicates the complex is formed between Sodium alginate and β-CD.

3.3.2. Swelling Ability:

The NSs appear to have the swelling capacity to assimilate water-exhibiting properties. A gel-like behavior because it happens for hydrogels. It is conceivable to assess the most extreme level of hydration by weighing the amount of water that can be retained by NS. The greatest level is called h and it is characterized as weight proportion H2O/NS⁴⁵. In arrange ponder, this esteem was found to be

● 1.239 for NS1,

● 1.00 for NS2,

● 2.08 for NS3 and

● 5.36 for NS4.

Among all NSs formulation NS2 and then NS1 has desirable swelling ability.

3.3.3. Solubility Studies:

Solubility Studies :

All NS subordinates were profoundly safe to natural solvents and they don't break down in water, diethyl ether, dichloromethane, chloroform, methanol, acetone, dmf, dmso, ethanol, ethyl acetic acid derivation which is demonstrated by the solvency test of Nanosponges.

3.4 Nanosponges parameters :

Table-4 : Mean Particle Size, PDI and Surface Charge of NSs

Formulation	Mean Particle size (nm)	Polydispersity index (PDI)	Zeta Potential (mV)
NS1	466 ± 18.2	0.36 ± 0.02	-31.5 ± 2.9
NS2	356 ± 1.4	0.28 ± 0.01	-10.4 ± 2.2
NS3	538 ± 29.8	0.32 ± 0.07	-38.2 ± 13.1
NS4	648 ± 34.9	0.39 ± 0.03	-43.6 ± 10.3

The mean particle difference across all prepared NSs were found to run from 350 to 650 nm in water. The molecule sizes of CD-NSs should be in the range between 250–600 nm. NSs in water have negative charge of the surface and run from −10 to −43 mV.

A least ± 20 mV zeta potential is needed for the physical safety of nanoparticle frameworks. In our study, the zeta potential of NS2 was found lower than −20 mV in water, which is a sign of stability and suitable preparation in a gastro-intestinal medium.

3.5 SEM Imaging of Nanosponges:

SEM images of prepared Nanosponge formulations are shown below.

Fig-9 :SEM of NS1 (1:1)

Fig-10 : SEM of NS2 (1:2)

Fig-11 :SEM of NS3 (1:4)

Fig-12 : SEM of NS4 (1:8)

SEM of β-Cyclodextrin Based Nanosponges appeared as cone-shaped molecules, Well-identified Nanosponges.

3.6 Simulated Biological Fluids determination:

3.6.1 Characterization of Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF):

Table-5 : pH, Osmolality And Buffer Capacity of SGF And SIF

Fluid names

Osmolality (mOsm/kg)

Buffer Capacity (μmol/mL/ΔpH)

Simulated Gastric fluid

1.2 ± 0.06

221 ± 0.04

17.4 ± 0.06

Simulated Intestinal fluid

7.2 ± 0.02

144 ± 0.05

12.6 ± 0.07

RANGE :

1. For Simulated Gastric Fluid:

● pH : 1.5-3.5

● Osmolality : 1 to 615 mOsm/kg

● Buffer Capacity : 7 - 18 μmol/mL/ΔpH

2. For Simulated Intestinal Fluid:

● pH : 6-7.4

● Osmolality : 35 to 631 mOsm/kg

● Buffer Capacity : 14 and 18 μmol/mL/ΔpH

In our study, pH, Osmolality and Buffer Capacity were found within the range, which is an indication of stable biological fluids.

3.6.2 Evaluation Of Simulated Saliva Fluid:

Table-6 : pH, relative density and viscosity of SSF

Fluid name

Relative Density

Viscosity (cP)

Simulated Saliva fluid

6.8 ± 0.02

1.001 ± 0.04

1.05 ± 0.08

RANGE : For Simulated Saliva Fluid

● pH : 6.6 - 7.1

● Relative Density : 1.004–1.009

● Viscosity : 1.05 cP and 1.29 cP

In our study, pH, Relative Density and Viscosity were found within the range, which is an indication of stable salivary fluid.

3.7 NS Stability In Biological Fluids:

3.7.1 Determination of stability in SGF on fresh, 3rd, 5th, 10th and 20th day:

Fig-13: Mean particle size estimation on distinctive days in SGF

Fig-14: Polydispersity Index estimation on distinctive days in SGF

Fig-15: Surface Charge estimation on distinctive days in SGF

3.7.2 Determination of stability in SIF on fresh, 3rd, 5th, 10th and 20th day

Fig-16: Mean particle size estimation on distinctive days in SIF

Fig-17: Polydispersity Index estimation on distinctive days in SIF

Fig-18: Surface charge estimation on distinctive days in SIF

Table-7 : Average parameters of NSs

Formulation	Mean Particle size (nm)	Polydispersity index (PDI)	Zeta Potential (mV)
NS1	438 ± 3.1	0.43 ± 0.06	-2.9 ± 1.8
NS2	301 ± 10..3	0.48 ± 0.02	-10.37 ± 2.4
NS3	435 ± 11.3	0.35 ± 0.05	+12.63 ± 8.2
NS4	489 ± 10.7	0.32 ± 0.01	-24.25 ± 11.6

In SGF, NS3 and NS4 were not stable inside 20 days owing to it may well be seen from molecule measure, PDI and Surface charge increment over time, Agreeing to the alter in molecule estimate information. The time subordinate cruel molecule measure variety of NS2 and NS1 was watched to be more constrained than NS3 and NS4.

NS2 and NS1 nano molecule sizes were obtained at stable level at the end of 20 day. NS4 were found to be not so stable in SIF for 20 days and their mean molecule measure, PDI and charge of surface enhanced significantly over time. In any case, though in general it was seen that the foremost suitable and stable formulation in GI media was NS2 taken after by NS1.

3.7.3 NS Stability in artificially prepared salivary fluid

Fig-19 : MPS OF NS IN SSF

Fig-20 : ZP Of NS In SSF

Fig-21 : PDI of SSF

Table-8 : NS parameters estimation in Artificially prepared Salivary Fluid

Formulation Code	Mean Particle Size (nm)	Zeta Potential (mV)	PDI
NS1	158 ± 2.4	-38.6 ± 1.4	0.824 ± 0.09
NS2	263 ± 0.02	+10.5 ± 0.65	0.303 ± 0.06
NS3	310 ± 1.8	+6.8 ± 0.87	0.684 ± 0.05
NS4	368 ± 2.2	-29.6 ± 0.34	0.396 ± 0.03

Within the amylase containing SSF, the zeta potential esteem of NS2 followed by NS3 are found to be suitable and stable. By observing all NSs, the existence of amylase in SSF has been observed to significantly influence the zeta potential esteem. Changed to positive charge due to a somewhat positive charge of the surface of amylase. Considerations were considered together when comparing stability of all formulations, NS2 followed by NS3 was found to be the significant and suitable formulation for oral route.

3.8 Adsorption Capacity Estimation of Phenol:

In different phenol concentration solutions the adsorption capacity estimation of Phenol:

At the conclusion of four hours of hatching period, the adsorption efficiency estimation of phenol by distinctive CD-NSs were measured in two diverse media that are artificially prepared Gastro and Intestinal fluids and are given within the figure below.

Table-9 : Adsorbed Phenol (%) In SGF

Phenol Concentration (ppm)	NS1 (%)	NS2 (%)	NS3 (%)	NS4 (%)
5	32 ± 0.01	61 ± 0.03	16 ± 1.01	12 ± 0.08
10	64 ± 0.06	70 ± 0.09	21 ± 0.04	8 ± 0.04
20	23 ± 0.03	46 ± 0.02	13 ± 0.09	15 ± 0.06
40	18 ± 0.08	57 ± 0.07	19 ± 0.04	7 ± 1.02

Fig-22 : Adsorbed phenol (%) in SGF

Table-10 : Adsorbed phenol (%) in SIF

Phenol Concentration (ppm)	NS1 (%)	NS2 (%)	NS3 (%)	NS4 (%)
5	46 ± 0.64	39 ± 0.05	22 ± 1.03	18 ± 0.24
10	63 ± 0.09	72 ± 0.16	27 ± 0.67	12 ± 1.08
20	36 ± 1.01	64 ± 0.43	31 ± 0.24	21 ± 0.02
40	12 ± 0.08	42 ± 0.04	33 ± 0.18	10 ± 0.03

Fig-23 : Adsorbed Phenol (%) In SIF

Adsorption efficiencies of Phenol in distinctive CD-NS preparations were estimated in two distinctive media that are in Simulated gastro and intestinal fluids. Then concluded by the end of 4- hours of hatching period. 70% in SGF and 72% in SIF has come-up as the highest adsorption of phenolic molecule p-Cresol from the artificial prepared in-vitro fluids by NS2-formulation. Then 64% in SGF and 63% in SIF of adsorption shown by NS1-formulation. Lastly, NS3-formulation is brought up by adsorbing 35% from the in-vitro artificially prepared fluids. Then NS4-formulation showed almost no or less adsorption in both the media.

4. CONCLUSION:

Sodium alginate (SA)-based β-cyclodextrin (β-CD) Nanosponges subsidiaries synthesized utilizing β-CD with diverse proportions of Sodium alginate as crosslinkers. They showed to be profitable in adsorption of the toxic molecule p-Cresol. NSs was found to be of 1:2 proportion of β-cyclodextrin with sodium alginate respectively as NS2-formulation with an adsorption efficiency of in-vitro phenol toxin is 70% in SGF and 72% in SIF which mimic biological fluids of the body and also exhibit promising approach in clearing poisonous compounds.

β-CD and Sodium alginate cross-linked NS was steady in synthetically prepared natural biological liquids. In addition, In vitro studies show the safety of these NSs for administering orally. In future, CD-NSs may be utilized to evacuate natural poisonous little particles and can come-up as an alternative treatment for CKD patients by reducing the dialysis frequency.

4.1 SCOPE:

● The present study gives the scope for another evaluations for characterization of CD-NS is ATIR

● Optimised formulation needs to be further studied for Toxicological estimations in simulated/small animal models

● Further long-term Stability studies need to be carried out as per the ICH - Guidelines.

5. REFERENCES:

1. Go, A.S., Chertow, G.M., Fan, D., McCulloch, C.E., Hsu, C.-Y., 2004. Chronic Kidney Disease and the Risks of Death, Cardiovascular Events, and Hospitalization. N. Engl. J. Med. 351, 1296–1305.

2. Hill, N.R., Fatoba, S.T., Oke, J.L., Hirst, J.A., O’Callaghan, C.A., Lasserson, D.S Hobbs, F.D.R., 2016. Global Prevalence of Chronic Kidney Disease – A Systematic Review and Meta-Analysis. PLoS ONE 11, e0158765.

3. Perlman, R.L., Finkelstein, F.O., Liu, L., Roys, E., Kiser, M., Eisele, G., Burrows-Hudson, S., Messana, J.M., Levin, N., Rajagopalan, S., Port, F.K., Wolfe, R.A., Saran, R., 2005. Quality of life in chronic kidney disease (CKD): a cross-sectional analysis in the Renal Research Institute-CKD study. Am. J. Kidney Dis. 45, 658–666.

4. GBD, 2017. Causes of Death Collaborators, 2018. Global, regional, and national age-sex- specific mortality for 282 causes of death in 195 countries and territories, 1980–2017: a systematic analysis for the Global Burden of Disease Study 2017. The Lancet 392, 1736–1788.

5. Kuehn BM. Excess Deaths From End-Stage Kidney Disease Early in Pandemic. JAMA. 2021;326(4):300.

6. Watanabe, Kimio; Watanabe, Tsuyoshi; Nakayama, Masaaki: Cerebro-renal interactions: Impact of uremic toxins on cognitive function. (2014) NeuroToxicology

7. Sun, C. Y.; Chang, S. C.; Wu, M. S.: Uremic toxins induce kidney fibrosis by activating intrarenal renin-angiotensin-aldosterone system associated epithelial-to-mesenchymal transition. (2012)

8. Vanholder, Raymond; Schepers, Eva; Pletinck, Anneleen; Nagler, Evi V.; Glorieux, Griet: The Uremic Toxicity of Indoxyl Sulfate and p-Cresyl Sulfate: A Systematic Review. (2014) Journal of the American Society of Nephrology

9. Opdebeeck, B.; Maudsley, S.; Azmi, A.; De, Maré A.; De, Leger W.; Meijers, B. et al.: Indoxyl Sulfate and p-Cresyl Sulfate Promote Vascular Calcification and Associate with Glucose Intolerance. (2019) Journal of the American Society of Nephrology

10. Koppe, L.; Pillon, N. J.; Vella, R. E.; Croze, M. L.; Pelletier, C. C.; Chambert, S. et al.: p-Cresyl sulfate promotes insulin resistance associated with CKD. (2013) Journal of the American Society of Nephrology

11. Wong, Jakk; Piceno, Yvette M.; DeSantis, Todd Z.; Pahl, Madeleine; Andersen, Gary L.; Vaziri, Nosratola D.: Expansion of urease- and uricase-containing, indole- and p-cresol-forming and contraction of short-chain fatty acid-producing intestinal microbiota in ESRD. (2014) American journal of nephrology

12. Sankowski, Bartłomiej; Księżarczyk, Karolina; Rackowska, Emilia; Szlufik, Stanisław; Koziorowski, Dariusz; Giebułtowicz, Joanna: Higher cerebrospinal fluid to plasma ratio of p-cresol sulfate and indoxyl sulfate in patients with Parkinson’s disease. (2020) Clinica Chimica Acta

13. Gryp T, Vanholder R, Vaneechoutte M, Glorieux G. p-Cresyl Sulfate. Toxins (Basel). 2017;9(2):52. Published 2017 Jan 29.

14. Ananya, K., Preethi, S., Amit, B.P., Gowda, D., 2020. Recent review on Nano sponge. Int. J. Res. Pharm. Sci. 11, 1085–1096.

15. Hu, C.-M.J., Fang, R.H., Copp, J., Luk, B.T., Zhang, L., 2013. A biomimetic nanosponge that absorbs pore-forming toxins. Nat. Nanotechnol. 8, 336–340. ISO 10993-5, 2009. Biological evaluation of medical devices —Part 5: Tests for in vitro cytotoxicity.

16. Bilensoy, E., Hincal, A.A., 2009. Recent advances and future directions in amphiphilic cyclodextrin nanoparticles. Expert. Opin. Drug Del. 6, 1161–1173.

17. Kurkov, S.V., Loftsson, T., 2013. Cyclodextrins. 453, 167–180.

18. Varan, G., Varan, C., Erdoğar, N., Hıncal, A.A., Bilensoy, E., 2017. Amphiphilic cyclodextrin nanoparticles. Int. J. Pharm. 531, 457–469.

19. Carrier, R.L., Miller, L.A., Ahmed, I., 2007. The utility of cyclodextrins for enhancing oral bioavailability. 123, 78-99.

20. Hirayama, F., Uekama, K., 1999. Cyclodextrin-based controlled drug release system. Adv. Drug Deliv. Rev. 36, 125–141.

21. Loftsson, T., Brewster, M.E., 2011. Pharmaceutical applications of cyclodextrins: Effects on drug permeation through biological membranes. J. Pharm. Pharmacol. 63, 1119–1135.

22. Uekama, K., Hirayama, F., Irie, T., 1998. Cyclodextrin drug carrier systems. Chem. Rev. 98, 2045–2076.

23. Swaminathan, S., Cavalli, R., Trotta, F., 2016. Cyclodextrin-based nanosponges: a versatile platform for cancer nanotherapeutics development. WIREs Nanomed. Nanobiotechnol. 8, 579–601.

24. Trotta, F., Zanetti, M., Cavalli, R., 2012. Cyclodextrin-based nanosponges as drug carriers. Beilstein. J. Org. Chem. 8, 2091–2099.

25. Torne, S.J., Ansari, K.A., Vavia, P.R., Trotta, F., Cavalli, R., 2010. Enhanced oral paclitaxel bioavailability after administration of paclitaxel-loaded nanosponges. Drug Delivery 17, 419–425.

26. Torne, S., Darandale, S., Vavia, P., Trotta, F., Cavalli, R., 2013. Cyclodextrin-based nanosponges: effective nanocarrier for Tamoxifen delivery. Pharm. Dev. Technol. 18, 619–625.

27. Swaminathan, S., Pastero, L., Serpe, L., Trotta, F., Vavia, P., Aquilano, D., Trotta, M., Zara, G., Cavalli, R., 2010. Cyclodextrin-based nanosponges encapsulating camptothecin: Physicochemical characterization, stability and cytotoxicity. Eur. J. Pharm. Biopharm. 74, 193–201.

28. Lembo, D., Swaminathan, S., Donalisio, M., Civra, A., Pastero, L., Aquilano, D., Vavia, P., Trotta, F., Cavalli, R., 2013. Encapsulation of Acyclovir in new carboxylated cyclodextrin-based nanosponges improves the agent's antiviral efficacy. Int. J. Pharm. 443, 262–272.

29. Darandale, S.S., Vavia, P.R., 2013. Cyclodextrin-based nanosponges of curcumin: formulation and physicochemical characterization. J. Incl. Phenom. Macro. 75, 315–322.

30. Swaminathan, S., Pastero, L., Serpe, L., Trotta, F., Vavia, P., Aquilano, D., Trotta, M., Zara, G., Cavalli, R., 2010. Cyclodextrin-based nanosponges encapsulating camptothecin: Physicochemical characterization, stability and cytotoxicity. Eur. J. Pharm. Biopharm. 74, 193–201.

31. Ansari, K.A., Vavia, P.R., Trotta, F., Cavalli, R., 2011. Cyclodextrin-Based Nanosponges for Delivery of Resveratrol. In Vitro Characterisation, Stability Cytotoxicity and Permeation Study. AAPS PharmSciTech 12, 279–286.

32. Cavalli, R., Akhter, A.K., Bisazza, A., Giustetto, P., Trotta, F., Vavia, P., 2010. Nanosponge formulations as oxygen delivery systems. Int. J. Pharm. 402, 254–257.

33. Mihailiasa, M., Caldera, F., Li, J., Peila, R., Ferri, A., Trotta, F., 2016. Preparation of functionalized cotton fabrics by means of melatonin loaded β-cyclodextrin nanosponges. Carbohydr Polym 142, 24–30.

34. Li, D., Ma, M., 1999. Nanosponges: From inclusion chemistry to water purifying technology. Chem. Tech. 29, 31–37.

35. Hu, C.-M.J., Fang, R.H., Copp, J., Luk, B.T., Zhang, L., 2013. A biomimetic nanosponge that absorbs pore-forming toxins. Nat. Nanotechnol. 8, 336–340. ISO 10993-5, 2009. Biological evaluation of medical devices —Part 5: Tests for in vitro cytotoxicity.

36. C. Varan, A. Anceschi, S. Sevli, N. Bruni, L. Giraudo, E. Bilgiç, P. Korkusuz, A.B. İskit, F. Trotta, E. Bilensoy, Preparation and Characterization of Cyclodextrin Nanosponges for Organic Toxic Molecule Removal, International Journal of Pharmaceutics (2020)

37. Preparation of Simulated Gastric Fluid, Bioscience Education [Internet], Available from :

38. https://bioscience-education.blogspot.com/2014/06/preparation-of-simulated-gastric-fluid.html?m=1, 20-June-2014.

39. Preparation of Simulated Intestinal Fluid, Bioscience Education [Internet], Available from:https://bioscience-education.blogspot.com/2014/06/preparation-of-simulated-intestinal.html?m=1 , 27-June-2014.

40. Electronic Supplementary Material (ESI) for Food & Function. This journal is © The Royal Society of Chemistry 2020. Available from : d0fo02319a1.pdf

41. Andi Sri, Suriati Amal et, al,. Preparation of Artificial Saliva Formulation, Research gate, October 2015.

42. Erika Stippler, Sabine Kopp, Jennifer B. Dressman et, al,. Comparison of US Pharmacopeia Simulated Intestinal Fluid TS (without pancreatin) and Phosphate Standard Buffer pH 6.8, TS of the International Pharmacopoeia with Respect to Their Use in In Vitro Dissolution Testing. May-2004.

43. Krstonošić, Milica Atanacković; Hogervorst, Jelena Cvejić; Mikulić, Mira; Gojković-Bukarica, Ljiljana. Development of HPLC method for determination of phenolic compounds on a core shell column by direct injection of wine samples. Acta Chromatographica, (), 1–5, Feb-2019.

: May 16- 2021.

45. Litou C, Vertzoni M, Goumas C, Vasdekis V, Xu W, Kesisoglou F, Reppas C. Characteristics of the human upper gastrointestinal contents in the fasted state under hypo- and A-chlorhydric gastric conditions under conditions of typical drug – drug interaction studies. Pharm Res 2016 Jun 14;33(6):1399–1412. Available from: http:// link.springer.com/10.1007/s11095-016-1882-8.

46. Crupi, V., Majolino, D., Mele, A., Melone, L., Punta, C., Rossi, B., Toraldo, F., Trotta, F., Venuti, V., 2014. Direct evidence of gel–sol transition in cyclodextrin-based hydrogels as revealed by FTIR-ATR spectroscopy. Soft Matter 10, 2320–2326.

47. N. M. Vageesh, Ramya Sri Sura, K Gulijar Begum, B Swathi. Formulation Development and In vitro Evaluation of Floating Tablets of Lafutidine by Employing Effervescent Technology. Asian J. Pharm. Res. 2017; 7(3): 189-197.

48. Shankar B. Kalbhare, Mandar J. Bhandwalkar, Rohit K. Pawar, Abhirup R. Sagre. Sodium Alginate cross-linked Polymeric Microbeads for oral Sustained drug delivery in Hypertension: Formulation and Evaluation. Asian J. Res. Pharm. Sci. 2020; 10(3):153-157.

49. Sampada V. Kadam, Nilima U. Rane, Chandrakant S. Magdum. Formulation and Evaluation of Orodispersible Tablets of Levamisole Hydrochloride. Asian J. Pharm. Tech. 2019; 9(2):63-68.

50. Priyanka V Dhalkar, Shivani S Jagtap, Suraj T Jadhav, Myuresh R. Redkar., Biradev S Karande. Formulation and Evaluation of in situ Gel Model Naproxen. Asian J. Pharm. Tech. 2019; 9(3):204-207.

51. Ashok Thulluru, S. Shakir Basha, C. Bhuvaneswara Rao, Ch. S. Phani Kumar, Nawaz Mahammed, K. Saravana kumar. Optimization of HPMC K100M and Sodium Alginate Ratio in Metronidazole Floating Tablets for the Effective Eradication of Helicobacter pylori. Asian J. Pharm. Tech. 2019; 9(3):195-203.

52. N. Sandhya Rani, M. Teja Krishna, V. Saikishore. . Design and Development of Sweet Potato Starch Blended Sodium Alginate Mucoadhesive Microcapsules of Glipizide. Research J. Pharma. Dosage Forms and Tech. 2012; 4(2): 119-123.

53. Megha B. Hiroji, Nagesh C., Devdatt Jani, Chandrashekhara S. Development and Evaluation of Transdermal Drug Delivery System using Natural Polysaccharides. Research J. Pharma. Dosage Forms and Tech. 2012; 4(5): 278-284.

54. LP Hingmire, VN Deshmukh, DM Sakarkar. LP Hingmire, VN Deshmukh, DM Sakarkar. Development and Evaluation of Sustained Release Matrix Tablets Using Natural Polymer as Release Modifier. Research J. Pharm. and Tech. 1(3): July-Sept. 2008; Page 193-196.

55. I Nyoman Suryadinata, Astina Putra, Tutiek Purwanti, Dewi Melani Hariyadi. Characterization of Microspheres in Alginate-Gelatin matrix with Ionotropic gelation method and Aerosolization Technique. Research J. Pharm. and Tech 2020; 13(9):4239-4243.

56. Sumit Kumar, Dinesh Chandra Bhatt. Influence of Sodium Alginate and Calcium Chloride on the Characteristics of Isoniazid Loaded Nanoparticles. Research J. Pharm. and Tech. 2021; 14(1):389-396.

Received on 20.11.2021 Modified on 08.04.2022

Asian J. Res. Pharm. Sci. 2022; 12(4):261-271.

DOI: 10.52711/2231-5659.2022.00045

Phenol (mg)	Dilution - I in Distilled H₂O	Dilution - II in Distilled H₂O	Phenol Solution Concentration
10 mg - Dilute in 10 ml	Withdrawn 1ml Dilute in 10ml	Withdrawn 0.5 ml and Dilute in 100 ml	5 ppm
10 mg - Dilute in 10 ml	Withdrawn 1ml Dilute in 10ml	Withdrawn 1 ml and Dilute in 100 ml	10 ppm
20 mg - Dilute in 10 ml	Withdrawn 1ml Dilute in 10ml	Withdrawn 1 ml and Dilute in 100 ml	20 ppm
40 mg - Dilute in 10 ml	Withdrawn 1ml Dilute in 10ml	Withdrawn 1 ml and Dilute in 100 ml	40 ppm