Evaluation of Antitumor Efficacy of Chitosan-Tamarind Gum Polysaccharide Polyelectrolyte Complex Stabilized Nanoparticles of Simvastatin

Figures & data

Table 1 Concentration of TGP (% w/v) and Ch (% w/v) for the Preparation of Nanoparticles

S. No.	TGP (%w/v)	Chitosan (%w/v)
1	0.03	0.03
2	0.03	0.01
3	0.03	0.02
4	0.01	0.03
5	0.01	0.01
6	0.01	0.02
7	0.02	0.03
8	0.02	0.01
9	0.02	0.02

Figure 1 Schematic representation of PEC formation between chitosan and tamarind gum polysaccharide.

Table 2 IR Interpretation of TGP

S.No.	Wave Numbers (cm^−1)	Functional Groups
1	3616	O-H Stretch
2	2361	COOH
3	2115	C≡C Stretch
4	1646	C=C Stretch
5	1042	C-O
6	1515	N-H Bend

Table 3 IR Interpretation of Simvastatin

S.No.	Wave Numbers (cm^−1)	Functional Groups
1	3645	N-H Stretch
2	2921	C-H Strech
3	1698	C≡O Stretch
4	1510	C=C Stretch
5	1384	C-H Bend
6	1271	C-N Bend
7	867	C-H Bend

Table 4 IR Interpretation of Chitosan

S.No.	Wave Numbers (cm^−1)	Functional Groups
1	3616	O-H Stretch
2	2361	COOH
3	2115	C≡C Stretch
4	1646	C=C Stretch
5	1042	C-O
6	1515	N-H Bend

Table 5 IR Interpretation of IR Spectra of the Physical Mixture of Ch, TGP and Simvastatin

S. No.	Wave Numbers (cm^−1)	Functional Groups
1	2361	COOH
2	1832	C-H Bend
3	1648	C=C Strech
4	1522	N-O Stretch
5	1108	C-O Stretch
6	758	C=C Bend

Figure 2 IR spectra of TGP.

Figure 3 IR spectra of simvastatin.

Figure 4 IR spectra of chitosan.

Figure 5 IR spectra of the physical mixture of Ch, TGP and simvastatin.

Figure 6 Surface plot for effect of independent variables over size of nanoparticles.

Figure 7 Surface plot for effect of independent variables over entrapment efficiency of nanoparticles.

Table 6 Characterization Parameters of PEC Stabilized Nanoparticles

Formulation	Characterization Parameters
	Physical Appearance	pH	Particle Size (nm)	Zeta Potential (mV)	Entrapment Efficiency (%)
T1	Clear solution	4.1±0.01	383.1	32.2	83.36± 0.23
T2	Clear solution	4.3±0.00	285.1	33.1	77.84±0.46
T3	Clear solution	4.3±0.01	306.0	32.6	73.22±0.32
T4	Clear solution	4.1±0.01	148.0	33.5	67.19±0.42
T5	Clear solution	4.1±0.01	53.3	33.4	71.01±0.36
T6	Clear solution	4.2±0.00	180.2	34.3	80.67±0.29
T7	Clear solution	4.2±0.01	103.3	35.2	82.29±0.23
T8	Clear solution	4.2±0.01	89.1	34.9	79.83±0.48
T9	Clear solution	4.2±0.01	73.3	33.6	75.54±0.46

Figure 8 SEM image of (A) Ch, (B) TGP, (C) drug precipitates and (D) nanoparticles-T5.

Figure 9 DR (%) of formulations via egg membrane (A) T1, T2, T3; (B) T4, T5, T6; and (C) T7, T8, T9.

Figure 10 DR (%) of formulations via tomato membrane (A) T1, T2, T3; (B) T4, T5, T6; and (C) T7, T8, T9.

Table 7 T_50% and T_80% of Prepared Nanoformulations

Formulation	Egg Membrane		Tomato Membrane
	T50%(min)	T80%(min)	T50%(min)	T80%(min)
T1	85	320	125	150
T2	210	310	105	150
T3	15	270	125	160
T4	90	270	120	150
T5	70	140	210	225
T6	23	150	120	150
T7	125	160	135	165
T8	125	160	110	140
T9	55	125	90	130

Table 8 Drug Release Data of PEC-based Nanoparticles Through Egg Membrane

Formulation	Kinetics
	Zero Order Kinetics		First Order Kinetics		Higuchi Kinetics		Baker Lonsdale Kinetics		Hixson-Crowell Kinetics		Kosermeyer Peppas Kinetics
	R^2	Ko	R^2	Ko	R^2	Ko	R^2	Ko	R^2	Ko	R^2	Ko	R^2
T1	0.389	0.043	0.103	0.0009	0.692	2.178	0.916	0.115	0.639	0.002	0.063	1.815	0.090
T2	0.5384	0.054	0.167	0.0011	0.726	1.482	0.895	0.092	0.642	0.003	0.010	2.207	0.095
T3	0.3537	0.041	0.091	0.0009	0.654	2.420	0.967	0.133	0.763	0.002	0.058	1.657	0.079
T4	0.3958	0.045	0.110	0.0011	0.707	2.096	0.907	0.112	0.642	0.003	0.073	1.893	0.073
T5	0.2541	0.043	0.094	0.0009	0.570	2.436	0.930	0.108	0.563	0.002	0.133	1.905	0.089
T6	0.2211	0.036	0.075	0.0011	0.540	2.930	0.965	0.112	0.559	0.002	0.153	1.676	0.123
T7	0.3614	0.045	0.108	0.0011	0.667	2.151	0.901	0.116	0.822	0.002	0.077	1.876	0.079
T8	0.3355	0.053	0.145	0.0011	0.646	1.716	0.925	0.060	0.783	0.003	0.129	2.795	0.067
T9	0.1942	0.045	0.108	0.0011	0.492	2.296	0.839	0.058	0.283	0.002	0.167	2.645	0.090

Table 9 Drug Release Data of PEC Based Nanoparticles Through Tomato Membrane

Formulation	Kinetics
	Zero-order Kinetics		First-order Kinetics		Higuchi Kinetics		Baker Lonsdale Kinetics		Hixson-Crowell Kinetics		Kosermeyer Peppas Kinetics
	R^2	Ko	R^2	Ko	R^2	Ko	R^2	Ko	R^2	Ko	R^2	Ko	R^2
T1	0.300	0.049	0.082	0.0009	0.617	1.920	0.925	0.071	0.656	0.003	0.103	1.637	1.638
T2	0.285	0.048	0.076	0.0009	0.605	1.978	0.946	0.066	0.640	0.002	0.123	1.606	1.606
T3	0.310	0.058	0.102	0.0011	0.616	1.489	0.750	0.028	0.625	0.003	0.145	2.067	2.068
T4	0.307	0.053	0.103	0.0009	0.625	1.719	0.927	0.044	0.663	0.003	0.133	1.989	1.990
T5	0.462	0.072	0.336	0.0018	0.639	1.139	0.522	0.011	0.673	0.003	0.402	5.869	5.869
T6	0.291	0.051	0.086	0.0009	0.609	1.848	0.934	0.055	0.641	0.003	0.127	1.746	1.746
T7	0.359	0.054	0.084	0.0009	0.780	1.641	0.928	0.057	0.696	0.003	0.088	1.808	1.808
T8	0.291	0.046	0.069	0.0009	0.608	2.054	0.953	0.073	0.695	0.002	0.084	1.482	1.482
T9	0.227	0.045	0.056	0.0006	0.536	2.244	0.953	0.072	0.470	0.002	0.164	1.407	1.408

Table 10 Particle Growth Analysis and Zeta Potential After 7th Day, 14th Day and 21st Day

Formulation	Particle Size Growth (nm)			Potential Difference (mV)
	After 7 Days	After 14 Days	After 21 Days	After 7 Days	After 14 Days	After 21 Days
T1	383.2	383.4	384.9	32.3	32.4	33.3
T2	285.1	285.3	287.3	33.2	33.4	34.9
T3	306.0	306.1	307.0	32.7	32.9	34.1
T4	148.1	148.6	150.1	33.6	33.8	34.8
T5	53.4	53.7	55.1	33.5	33.8	34.6
T6	180.2	180.3	181.9	34.4	34.6	36.1
T7	103.5	103.6	105.8	35.2	35.5	36.8
T8	89.2	89.5	91.1	35.1	35.2	37.0
T9	73.4	73.7	74.9	33.6	35.7	35.2

Table 11 Stability Study Parameters of Nanoformulations

Formulation	Particle Size (nm)		Entrapment Efficiency (%)
	Initial Day	After 45 Days	Initial Day	After 45 Days
T1	383	387	83.36±0.42	66.89±0.14
T2	285	289	77.84±0.46	77.02±0.29
T3	306	310	73.22±0.32	73.18±0.42
T4	148	155	67.19±0.23	82.99±0.26
T5	53	59	71.01±0.36	70.16±0.19
T6	180	186	80.67±0.29	79.21±0.46
T7	103	106	82.29±0.23	81.89±0.37
T8	89	94	79.83±0.48	79.12±0.25
T9	73	78	75.54±0.46	74.67±0.38

Table 12 List of Chitosan-based PEC Nanoparticles and Their Outcomes

Chitosan-based PEC Nanoparticles	Outcome
Attallah et al prepared pectin/chitosan/jasmine oil nanoparticles (Pec/CS/JO NPs) by using Box-Behnken design (BBD).^Citation81	The outcome was that pec/CS/JO NPs was effective against (MSF-7) breast cancer cells, which was evaluated by using MTT assay, where it was found that normal cells were not affected by the prepared nanoparticle.^Citation81
Guo et al formulated a chitosan-based nanogel to inhibit the proliferation of breast cancer cell lines, where the model drug of 10-hydroxycamptothecin (HCPT) was entrapped into core by facile diffusion to form chitosan/10-hydroxycamptothecin (CS/HCPT).^Citation82	From the prepared formulation the outcome was evaluated by assessing particle size, drug loading content and drug loading efficiency. Further cytotoxicity, apoptosis of CS/HCPT, was also investigated in vitro.^Citation82
Nascimento et al synthesized a delivery system in which non-targeted delivery was done of chitosan, polyethylene glycol and siRNA.^Citation83	The outcome showed that there was an inhibition in tumor tissues in animal model and faster elimination.^Citation83
Nascimento et al did another study and synthesized another delivery system for targeted delivery, which was done by chitosan, polyethylene glycol, RGFR-targeting peptide and siRNA.^Citation84	In this research combination therapy was used, where chitosan-based nanoparticles were combined with cisplatin in cancer models and tumor tissues.^Citation84
Jain et al developed a chitosan nanoparticle which was coupled with hyaluronic acid with the help of inotropic gelation method, where 5-fluorouracil (5FU) was chosen as the drug and used for the colon cancer treatment.^Citation85	In the outcome, HA was incorporated in the chitosan nanoparticles drug delivery system due to an elevated level of HA receptors around tumor tissues. So, in the result, nanoparticles can target colon tumors via both EPR effect and binding of hyaluronic acid to HA receptors and chitosan-based nanoparticles were already loaded with 5-fluorouracil drug.^Citation85
Zhang et al reported a design of pH-mediated chitosan-based microgel DDS for treatment of cancer, where chitosan powder was dissolved in water and then it was reacted with Glycidyltrimethylammonium chloride to form N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC). Further nanoparticles of HTCC were fabricated by ionotropic method.^Citation86	Methotrexate disodium was also loaded in HTCC and in the outcome of this research, it was found that in cell experiments MTX-loaded HTCC highest mortality rate of Hela cells and cancerous cells was found as compared to the pure drug group and non-conjugated MTX- chitosan nanoparticle group.^Citation86
Yang et al successfully formulated oxaliplatin and miRNA-205-5p loaded polyethyleneimine (PEI)/hyaluronic acid (HA) mesoporous silica-based nanoparticles (OXmi-HSMN). This formulation was made to improve therapeutic efficacy in colon cancers.^Citation87	In the research, when tumors were extracted from the mice and processed for TUNEL assay, it can clearly be seen in the assay that OXmi-HMSN encouraged apoptosis of tumor cells when compared to any other group which is producing a superior anticancer effect. OXmi-HMSN leads to inhibition of tumor growth which is greater than any other group, either OXL or OXL-MSN. So, their study provides the dual therapeutics for enhanced therapeutic efficacy in the treatment of colon cancer.^Citation87
Cui et al did research and formulated a T7-modified, magnetic PLGA nanoparticulate system (MNP/T7-PLGA NPs) through the process of single-emulsion solvent evaporation method, in which nanoparticles which are magnetic hydrophobic (MNPs) were entrapped into PLGA NPs.^Citation88	In the outcome of this research, when comparison was done between other conventional magnetic nanoparticles and these PLGA NPs, it was found that PLGA NPs are more beneficial due to their capability of co-encapsulation of various drugs. The efficiency of treatment was investigated both in vitro and in vivo.^Citation88

Figure 11 Anticancerous activity of nanoparticles (T5) and pure drug precipitates.

Figure 12 Bright field image of (A) untreated MCF-7 cells, (B) MCF-7 cells treated with nanoparticles (T5) and (C) MCF-7 cells treated with pure simvastatin.

Table 13 Cell Death (%) After Administration of Pure Drug and Nanoparticles at Different Concentrations

Concentration (µg/mL)	Cell Death (%)
	Pure Drug	Nanoparticle (T5)
0	0±0	0±0
2	−10.61±0.37	−1.36±0.96
4	−12.24±0.65	−2.1±1.74
8	−20.75±0.84	−3.12±1.87
10	−32.86±0.97	−3.9±2.01

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