Metformin Hydrochloride Loaded Mucoadhesive Microspheres and Nanoparticles for Anti-Hyperglycemic and Anticancer Effects Using Factorial Experimental Design

Figures & data

Table 1 Formulations Protocol for Microsphere and Nanoparticles

Batch No	Batch Size	Drug Metformin HCl (g)	Eudragit RSPO (g)	Drug Loading (%)
A1	1 g	0.5	0.5	50%
A2	2 g	1.5	0.5	75%
A3	2 g	0.5	1.5	25%
A4	3 g	1.5	1.5	50%
B1	1 g	0.5	0.5	50%
B2	2 g	1.5	0.5	75%
B3	2 g	0.5	1.5	25%
B4	3 g	1.5	1.5	50%

Figure 1 Schematic diagram of drug permeation–pharmacodynamics correlation. Using this schematic diagram, a simple correlation between in vitro pharmacokinetic and in vivo pharmacodynamic study had been tried in this research.

Table 2 The Effects of Polymeric Concentration, Drug Loading in Total Drug-Polymer Amount on Particle Size

Formulation	Batch no.	Theoretical Drug Loading in Total Amount of Drug Polymer	Particle Size (Mean ± SD)	Polydispersity Index (PDI)
Microsphere	A1	50% (1g)	5.28 ± 0.72 µm	0.40
	A2	75% (2g)	5.53 ± 0.64 µm	0.28
	A3	25% (2g)	2.42 ± 0.93 µm	0.25
	A4	50% (3g)	4.11 ± 1.10 µm	0.15
Nanoparticles	B1	50% (1g)	160.10 ± 19.07 nm	0.10
	B2	75% (2g)	230.10 ± 37.31 nm	0.31
	B3	25% (2g)	359.90 ± 63.08 nm	0.10
	B4	50% (3g)	254.90 ± 37.63 nm	0.23

Notes: Four batches of microspheres (A1, A2, A3 and A4) and four batches of nanoparticles (B1, B2, B3 and B4).

Figure 2 Z-average (nm) value and PDI of Metformin microspheres of batch (a) A1, (b) A2, (c) A3 and (d) A4 and nanoparticles of batch (e) B1, (f) B2, (g) B3 and (h) B4.

Figure 3 SEM images of metformin microspheres of batch (a) A1, (b) A2, (c) A3 and (d) A4, and nanoparticles of batch (e) B1, (f) B2, (g) B3 and (h) B4.

Figure 4 DSC thermogram of all batches of Metformin HCl loaded microspheres (a) and nanoparticles (b) in relation to pure Metformin HCl corresponding to the solid red line in both images. (a) Four batches of microspheres (A1, A2, A3 and A4 designated by solid purple, green, blue and black line respectively) and (b) four batches of nanoparticles (B1, B2, B3 and B4 designated by solid green, black, purple and blue line respectively).

Table 3 FTIR Vibrational Band Assignment of Pure Metformin HCl API

Compound	Tentative Assignment	Frequency (cm⁻¹)
Metformin HCl	Primary N-H stretching	3372 (sm)
	Asymmetric N-H stretching	3300 (m)
	Symmetric N-H stretching	3176 (m)
	(CH3)2 N absorption	2619 (vw) - 2816 (w)
	Primary N-H deformation	1583 (s)- 1626 (s)
	Asymmetric N-H deformation	1566 (s)
	Symmetric N-H deformation	1475 (sm)
	N-H deformation	1417 (m)- 1455 (m)
	C-N stretching	1033 (w)-1170(w)
	C-N-C deformation	418–580
	C-H out of plane bending	633 (m)
	N-H out of plane bending	936 (mw)
	NH2 rocking	800 (w)
	N-H wagging	736 (mw)

Figure 5 FTIR spectrum of pure metformin HCl powder corresponding to the solid green line (a), all batches metformin HCl loaded microspheres (b) and nanoparticles (c). Four batches of microspheres (A1, A2, A3 and A4 designated by solid Orange, blue, green and purple line respectively) and four batches of nanoparticles (B1, B2, B3 and B4 designated by solid purple, pink, red and green line respectively).

Table 4 Calculation of Drug Loading for Metformin HCl Microspheres and Nanoparticles

Batches	Theoretical Drug Loading	Practical Drug Loading	Drug Entrapment Efficiency (EE%)
A1	50%	20.45%	40.94%
A2	75%	50.1%	66.78%
A3	25%	11.48%	45.9%
A4	50%	42.595%	85.19%
B1	50%	20.9%	41.8%
B2	75%	25.11%	33.48%
B3	25%	16.86%	67.43%
B4	50%	22.07%	44.14%

Notes: Four batches of microspheres (A1, A2, A3 and A4) and four batches of nanoparticles (B1, B2, B3 and B4).

Figure 6 Comparison of cumulative % release in pH 6.8 phosphate buffer of metformin HCl from microspheres and nanoparticles prepared by emulsion solvent evaporation and nanoprecipitation technique respectively, where four formulations of metformin HCl loaded microspheres and nanoparticles indicated to (a and e) for zero order release profile, (b and f) for first order release profile, (c and g) for the Higuchi model and (d and h) for the Korsmeyer–Peppas model respectively. For all microsphere drug release figures (a-d), solid blue, Orange, ash and black line indicates microsphere batch A1, A2, A3 and A4 respectively whereas for all nanoparticles drug release figures (e-h), solid purple, red, yellow and green line indicates nanoparticle batch B1, B2, B3 and B4 respectively.

Table 5 Summary of Mathematical Kinetics Test of Prepared Formulations

Basic Buffer		Microspheres				Nanoparticles
		A1	A2	A3	A4	B1	B2	B3	B4
Zero order	K0	11.581	10.300	9.441	12.807	5.292	6.029	8.293	7.256
	R^2	0.6034	0.9018	0.9143	0.0956	0.9278	0.8130	0.9618	0.8699
First order	K1	0.332	0.210	0.172	0.632	0.068	0.082	0.124	0.104
	R^2	0.9571	0.9418	0.9026	0.9388	0.9344	0.8787	0.8986	0.8530
Higuchi	Kh	32.967	28.689	26.145	37.071	13.936	16.130	21.554	19.115
	R^2	0.9624	0.9617	0.9161	0.8380	0.8892	0.9292	0.8353	0.8340
Korsmeyer-Peppas	Kkp	37.979	21.356	16.835	58.283	8.009	13.941	7.978	10.897
	R^2	0.9686	0.9824	0.9511	0.9742	0.9417	0.9283	0.9577	0.9135
	n	0.427	0.650	0.723	0.264	0.793	0.578	1.019	0.797

Table 6 Best Fitted Models for Optimized Formulations

Formulations	Best Fitted Model	n value	Release Mechanism
A1	Korsmeyer-Peppas	0.427	Fickian (case I) diffusion
A2	Korsmeyer-Peppas	0.650	Anomalous / non – Fickian transport
A3	Korsmeyer-Peppas	0.723	Anomalous / non – Fickian transport
A4	Korsmeyer-Peppas	0.264	Fickian (case I) diffusion
B1	Korsmeyer-Peppas	0.793	Anomalous / non – Fickian transport
B2	Korsmeyer-Peppas	0.578	Anomalous / non – Fickian transport
B3	Korsmeyer-Peppas	1.019	Super case II transport
B4	Korsmeyer-Peppas	0.797	Anomalous / non – Fickian transport

Figure 7 Permeability profiles of metformin HCl API powder (MAPI) indicated by solid red line, selected formulation A4 of microspheres (A4MS) indicated by solid blue line and B3 of nanoparticles (B3NP) indicated by solid green line (n = 3).

Figure 8 The apparent permeability, Papp (cm/s x 10–5) of metformin (mucosal -to- serosal) from metformin HCl API powder (MAPI), selected formulation A4 of microspheres (A4MS) and B3 of nanoparticles (B3NP) (n = 3).

Table 7 Effect of Metformin HCl API Powder (MAPI), Selected Formulation A4 of Microspheres (A4MS) and B3 of Nanoparticles (B3NP) on Blood Sugar Level in Mice Model

Groups	FBS (mmol/L)	OGTT (mmol/L)
		Zero Hour	1st Hour	2nd Hour	3rd Hour	6th Hour
NC	5.02 ± 2.32	7.33 ± 0.40	7.08 ± 0.15	6.83 ± 2.19	4.97 ± 1.28	4.20 ± 0.95
MAPI	4.38 ± 1.56	7.47 ± 0.41	6.72 ± 0.73	6.25 ± 1.71	4.40 ± 1.01	3.62 ± 1.00
A4MS	5.18 ± 1.24	7.58 ± 0.5	4.15 ± 0.98*	3.72 ± 0.89	3.00 ± 0.79	2.18 ± 0.44
B3NP	4.20 ± 2.37	7.55 ± 0.38	4.91 ± 0.47*	4.61 ± 0.57	3.72 ± 0.48	2.87 ± 0.42

Notes: All data are given as mean ± SD for six animals in each group. Statistically significant results are indicated as (*) p < 0.05 versus untreated negative control group (NC).

Figure 9 Point to point correlation between the in-vitro pharmacokinetic (drug permeation profiles) and its in-vivo pharmacodynamic effect (Blood glucose reduction) of metformin HCl API (MAPI), metformin HCl loaded microsphere (A4MS) and nanoparticles (B3NP) whereas, Cumulative % drug permeation is indicated by blue points and reduction of Blood glucose concentration is indicated by Orange points.

Figure 10 Correlation of the pharmacokinetic (drug permeation profiles) and its pharmacodynamic (hypoglycemic activity) effect: metformin HCl API (MAPI), metformin HCl loaded microsphere (A4MS) and nanoparticles (B3NP).

Table 8 Effect of Metformin HCl Nanoparticles on Cancerous HeLa Cell Line

Samples	Percentage of Survival HeLa Cells
Solvent -	100%
Solvent +	More than 95%
B2A	10%- 20% (Cytotoxic to cancer cells)
B3A	10%- 20% (Cytotoxic to cancer cells)
B4A	10%- 20% (Cytotoxic to cancer cells)
B2B	More than 95% (no anticancer property)
B3B	10%- 20% (Cytotoxic to cancer cells)
B4B	20%- 30% (Cytotoxic to cancer cells)

Notes: HeLa cells were cultured with different samples, such as with (Solvent +) or without (Solvent-) solvent, 500µg/mL concentration of B2, B3, and B4 formulations marked as B2A, B3A and B4A respectively and 250µg/mL concentration of B2, B3, and B4 formulations named as B2B, B3B and B4B, respectively of metformin loaded nanoparticles.

Figure 11 Effect of Metformin HCl Nanoparticles on cancerous HeLa cell line. HeLa cells were cultured with different samples, such as without solvent(a) or with solvent (b), 500 µg/mL concentration of B2, B3, and B4 formulations marked as B2A (c), B3A (e) and B4A (g), respectively and 250 µg/mL concentration of B2, B3, and B4 formulations named as B2B (d), B3B (f) and B4B (h), respectively of metformin loaded nanoparticles.