The fate of calcium carbonate nanoparticles administered by oral route: absorption and their interaction with biological matrices

Figures & data

Table 1 Physicochemical properties of the calcium carbonates examined

Characterization		Samples
Instrument	Physicochemical parameters	B-Cal	N-Cal
XRD	Crystalline phase	Calcite	Calcite
	Crystallite size	~40 nm	~30 nm
FT-IR	Major bands	710 cm^−1: CO3^2− deformation (sym)
		875 cm^−1: CO3^2− deformation (asym)
		1,440 cm^−1: CO3^2− stretching (asym)
		2,500–3,000 cm^−1: –CH2–stretching	–
	Chemical property	Calcite with organic moiety	Calcite
SEM	Primary particle size	~40 nm	~30 nm
	Particle size distribution	Inhomogeneous	Fairly homogeneous
DLS	Z-average (hydrodynamic size)	7,363 nm	2,187 nm
	PDI	0.698	0.307
BET	Specific surface area	0.83 m^2/g	15.8 m^2/g
LSE	Zeta potential	−2.98 mV	+16.37 mV

Abbreviations: asym, asymmetric; B-Cal, bulk calcium carbonates; BET, Brunauer-Emmett-Teller nitrogen adsorption-desorption; DLS, dynamic light scattering; FT-IR, Fourier transform infrared; LSE, light scattered electrophoresis; N-Cal, nano calcium carbonates; PDI, polydispersity index; SEM, scanning electron microscopy; sym, symmetric; XRD, X-ray diffraction.

Figure 1 XRD patterns and SEM images of B-Cal (a) and N-Cal (b) as produced (A) and after being incubated in simulated gastric (B) and intestinal fluids (C) and plasma (D).

Notes: (hkl) Miller indexing in XRD patterns (A) are typical patterns of the calcite phase (JCPDS No 47-1743); Asterisks in XRD patterns stand for the evolution of dicalcium phosphate dihydrate (JCPDS No 72-0714); SEM images were obtained after incubation for 1 hour in simulated gastric fluid and 4 hours in simulated intestinal fluid or plasma condition.

Abbreviations: arb, arbitrary; B-Cal, bulk calcium carbonates; hkl, Miller indexing; JCPDS, Joint Committee on Powder Diffraction Standards; N-Cal, nano calcium carbonates; SEM, scanning electron microscopy; XRD, X-ray diffraction; h, hours.

Table 2 In vitro and ex vivo dissolution and protein fluorescence quenching ratio of calcium carbonate materials

Condition	Time (h)	In vitro				Ex vivo
		Dissolution (%)		Quenching ratio (%)		Dissolution (%)		Quenching ratio (%)
		B-Cal	N-cal	B-Cal	N-cal	B-Cal	N-cal	B-Cal	N-cal
Gastric	0.25	0.75	0.73	23.98	81.96	2.14	3.59	16.28	83.97
	0.5	0.75	0.74	32.63	86.21	1.69	3.39	28.08	89.58
	1	0.75	0.82	43.55	86.65	1.66	3.37	31.98	92.15
Intestinal	0.5	<0.01	<0.01	21.93	76.66	<0.01	<0.01	25.15	81.05
	1	<0.01	<0.01	25.86	81.22	<0.01	<0.01	23.62	85.71
	4	<0.01	<0.01	27.99	82.88	<0.01	<0.01	52.57	89.55
Plasma	0.5	<0.01	<0.01	7.97	28.23	<0.01	<0.01	46.83	90.14
	1	<0.01	<0.01	18.49	68.92	<0.01	<0.01	53.67	93.29
	4	<0.01	<0.01	24.53	80.25	<0.01	<0.01	60.53	94.49

Abbreviations: B-Cal, bulk calcium carbonates; N-Cal, nano calcium carbonates; h, hours.

Table 3 List of the most abundant plasma proteins bound on B-Cal and N-Cal as determined by LC–MS

MW	pI	B-Cal (spot number)	No	N-Cal (spot number)	pI	MW
25	5.7	Ig light chain (19)	1	Ig light chain (19)	5.7	25
70	5.4	Prothrombin (3)	2	Apolipoprotein A-I preprotein (23)	5.5	30
55	4.6	Fetuin A (10)	3	Apolipoprotein E (22)	5.5	36
29	4.8	Apolipoprotein D (18)	4	Prothrombin (3)	5.4	70
11	4.6	Apolipoprotein C-III (24)	5	Apolipoprotein A-IV precursor (13)	5.3	44
50	5.6	Fibrinogen gamma chain precursor (7)	6	Apolipoprotein C-III (24)	4.6	11
44	5.3	Apolipoprotein A-IV precursor (13)	7	Ig heavy chain (mu) (2)	6.0	72
58	5.4	Ig heavy chain (alpha) (8)	8	Fibrinogen gamma chain precursor (7)	5.6	50
72	6.0	Ig heavy chain (mu) (2)	9	Fetuin A (10)	4.6	55
48	4.7	Alpha-2 acid-glucoprotein (14)	10	Alanine aminotransferase 1 (9)	5.2	55
65	5.6	Albumin (4)	11	Ig heavy chain (alpha) (8)	5.4	58
42	6.5	Fetuin B (16)	12	Fetuin B (16)	6.5	42
60	6.7	Fibrinogen alpha chain isoform 1 precursor (5)	13	Fibrinogen alpha chain (1)	5.7	95
42	5.2	Actin, cytoplasmic 2 (12)	14	Apolipoprotein D (18)	4.8	29
67	6.9	C4b-binding protein alpha chain (6)	15	Fibrinogen alpha chain isoform 1 precursor (5)	6.7	60
51	6.2	P55 protein (fragment) (20)	16	C4b-binding protein alpha chain (6)	6.9	67
531		Plectin isoform (fragment) (17)	17	P55 protein (fragment) (20)	6.2	51
37		UDP-glucuronosyltransferase 2B17-like isoform X3 (15)	18	Unnamed protein (21)		30

Note: Proteins marked by bold font were found in B-Cal and N-Cal.

Abbreviations: B-Cal, bulk calcium carbonates; Ig, immunoglobulin; LC–MS, liquid chromatography–mass spectrometry; MW, molecular weight; N-Cal, nano calcium carbonates; No, number; pI, isoelectric point.

Figure 2 Plasma protein binding profiles of calcium carbonates separated by one-dimensional (A), and two-dimensional (B), gel electrophoresis.

Abbreviations: B-Cal, bulk calcium carbonates; N-Cal, nano calcium carbonates.

Table 4 Biokinetic parameters and absorption of calcium carbonates after oral administration to rats

	B-Cal				N-Cal
	Single dose		14-day repeated dose		Single dose		14-day repeated dose
	Male	Female	Male	Female	Male	Female	Male	Female
Cmax (μg/mL)	18.0±3.8a,b	14.0±1.9a	16.2±2.4a	17.3±3.4a,b	22.8±3.6c	21.5±3.4b,c	20.2±2.0b	22.0±3.5b
Tmax (h)	2	2	2	2	1	1	1	1
AUC (h × μg/mL)	68.4±11.5	64.6±4.9	71.8±4.3	69.4±9.4	64.1±7.2	64.0±11.8	66.1±6.1	61.8±8.5
T1/2 (h)	2.7±0.4	3.0±0.6*	3.2±0.7b	3.7±0.1b,*	2.5±0.7	2.5±1.2	1.9±0.3a	2.0±0.8a
MRT (h)	4.6±1.3	4.8±0.7	5.1±0.7b	5.5±0.5b	4.1±1.3	4.2±2.1	4.2±0.3a	3.4±0.6a
Absorption (%)d	4.6±0.8	4.3±0.3	4.8±0.3	4.6±0.6	4.3±0.5	4.3±0.8	4.4±0.4	4.1±0.6

Notes:

a,b,c Significant difference among four groups (male, female, single, or 14-day repeated dose) administered B-Cal or N-Cal;

* Significant difference between single and repeated dose groups;

d Absorption was calculated based on AUC values.

Abbreviations: AUC, area under the plasma concentration-time curve; B-Cal, bulk calcium carbonates; C_max, maximum concentration; MRT, mean residence time; N-Cal, nano calcium carbonates; T_1/2, half-life; T_max, time to maximum concentration.

Figure 3 Plasma concentration-time curves (A–D) and tissue distribution (E, F) of calcium carbonates after a single (A, B), or 14 days of daily oral treatment (C–F) to rats.

Notes: Biokinetic data are presented as increase in calcium levels after subtracting the basal plasma calcium levels observed in untreated controls; (A) Biokinetics after administering a single dose to male rats; (B) Biokinetics after administering a single dose to female rats; (C) Biokinetics after 14-days of daily oral treatment in male rats; (D) Biokinetics after 14-days of daily oral treatment in female rats; (E) Tissue distribution in male rats; (F) Tissue distribution in female rats. (a) and (b) is used when there is no significant difference between two groups (control and N-Cal/N-Cal and B-Cal), but significant difference is found when three groups (control, N-Cal, B-Cal) are compared.

Abbreviations: B-Cal, bulk calcium carbonates; N-Cal, nano calcium carbonates.

Figure S1 XRD patterns (A) and FT-IR spectra (B) of (a) B-Cal and (b) N-Cal.

Abbreviations: arb, arbitrary; B-Cal, bulk calcium carbonates; FT-IR, Fourier transform infrared; N-Cal, nano calcium carbonates; XRD, X-ray diffraction.

Figure S2 SEM images for the produced B-Cal (A, C) and N-Cal (B, D).

Abbreviations: B-Cal, bulk calcium carbonates; N-Cal, nano calcium carbonates; SEM, scanning electron microscopy.

Figure S3 Hydrodynamic size distributions of B-Cal (A), and N-Cal (B) in aqueous suspension as determined by dynamic light scattering.

Abbreviations: B-Cal, bulk calcium carbonates; N-Cal, nano calcium carbonates; PDI, polydispersity index

Figure S4 Zeta potential distributions of B-Cal (A), and N-Cal (B).

Abbreviations: arb, arbitrary; B-Cal, bulk calcium carbonates; N-Cal, nano calcium carbonates.

Figure S5 XRD patterns of B-Cal (a), and N-Cal (b), incubated in simulated body fluids: gastric fluid (A), intestinal fluid (B), and plasma (C) after 24 or 48 hours.

Note: Asterisks indicate the evolution of dicalcium phosphate dihydrate (JCPDS No 72-0714).

Abbreviations: arb, arbitrary; B-Cal, bulk calcium carbonates; JCPDS, Joint Committee on Powder Diffraction Standards; N-Cal, nano calcium carbonates; XRD, X-ray diffraction; h, hours.

Figure S6 XANES at calcium K-edge for B-Cal (A), and N-Cal (B), treated with simulated body fluid, untreated (a) and gastric fluid (b), intestinal fluid (c) and plasma (d) for 48 hours.

Note: The inset table shows the edge position obtained by secondary differentiation of peak.

Abbreviations: arb, arbitrary; B-Cal, bulk calcium carbonates; N-Cal, nano calcium carbonates; XANES, X-ray absorption near edge structure.

Figure S7 XRD patterns of B-Cal (a) and N-Cal (b) incubated ex vivo in fluids extracted from tissues: gastric fluid (A), intestinal fluid (B), and plasma (C).

Note: Asterisks indicate the evolution of dicalcium phosphate dihydrate (JCPDS No 72-0714).

Abbreviations: arb, arbitrary; B-Cal, bulk calcium carbonates; JCPDS, Joint Committee on Powder Diffraction Standards; N-Cal, nano calcium carbonates; XRD, X-ray diffraction; h, hours.

Figure S8 SEM images of B-Cal (a) and N-Cal (b) after incubation ex vivo in tissue extracted fluids: gastric fluid for 1 hour (A), intestinal fluid for 4 hours (B), and plasma for 4 hours (C).

Abbreviations: B-Cal, bulk calcium carbonates; N-Cal, nano calcium carbonates; SEM, scanning electron microscopy.

Figure S9 XRD patterns of B-Cal (a), and N-Cal (b), obtained from stomach (A), and intestine (B) following oral administration to rats.

Note: Asterisks and triangles indicate dicalcium phosphate dihydrate (JCPDS No 72-0714) and calcite, respectively.

Abbreviations: arb, arbitrary; B-Cal, bulk calcium carbonates; JCPDS, Joint Committee on Powder Diffraction Standards; N-Cal, nano calcium carbonates; XRD, X-ray diffraction; h, hours.

Figure S10 SEM images of B-Cal (a) and N-Cal (b) obtained from stomach at 1 hour postadministration (A), and intestine at 4 hours postadministration (B).

Abbreviations: B-Cal, bulk calcium carbonates; N-Cal, nano calcium carbonates; SEM, scanning electron microscopy.