CD-340 functionalized doxorubicin-loaded nanoparticle induces apoptosis and reduces tumor volume along with drug-related cardiotoxicity in mice

Figures & data

Table 1 Composition of various experimental nanoparticle formulations with their physicochemical characteristics

Formulation	PLGA:Drug: DCM (Ratio by weight)	Sabilizer used	Average particle diameter (Z-average) (nm)	Polydispersity index^a	Zeta potential (mV)	%Drug loading^a	%Entrapment efficiency^a
PVA	40:4:1	PVA (2.5% w/v)	357	0.398±0.03	−7.39±0.008	5±0.02	55%±0.3
PVA+TWEEN	40:4:1	PVA (2.5% w/v) &Tween 80 (14% v/v) (F2)	220	0.234±0.01	−14.7±0.009	8.5±0.015	93.5%±0.21
PVA+PLU-68	40:4:1	PVA (2.5% w/v) & Pluronic-68 (0.5% w/v)	449	0.041±0.018	−12.2±0.01	7±0.12	77%±0.5
PVA+PLU-127	40:4:1	PVA (2.5% w/v) & Pluronic- 127 (0.5% w/v)	466	0.419±0.022	−13.7±0.011	6.9±0.03	75%±0.33
PVA+TPGS	40:4:1	PVA (2.5% w/v) & TPGS (0.03%w/v)	640	0.925±0.011	−9.98±0.008	6.03±0.15	66%±0.83
Antibody- conjugated formulation	40:4:1	PVA (2.5% w/v) &Tween 80 (14% v/v)	241	0.230±0.009	−2.93±0.013	8±0.01	88%±0.17

Note: ^aEach value represents mean ± SD (n=3).

Abbreviations: PLGA, poly (lactide-co-glycolide); DCM, dichloromethane; PVA, poly(vinyl alcohol); TWEEN, tween 80; PLU-68, pluronic F68; PLU-127, pluronic F127; TPGS, D -α-tocopherol polyethylene glycol succinate.

Figure 1 AFM and TEM images of experimental nanoparticles.

Notes: AFM (A) DOX-NP, (B) DOX-Ab-NP and TEM (C) DOX-NP and (D) DOX-Ab-NP.

Abbreviations: PLGA, polylactic-co-glycolic acid; PVA, poly (vinyl alcohol); AFM, Atomic force microscopy; DOX, doxorubicin; Ab, antibody; NP, nanoparticle; TEM, transmission electron microscopy.

Table 2 Weight % and atomic % of elements in various nanoparticles

Sample	CK		OK		NK		NaK		SK
	Weight %	Atomic %	Weight %	Atomic %	Weight %	Atomic %	Weight %	Atomic %	Weight %	Atomic %
BL-NP	42.13	49.64	56.86	50.29	-	-	-	-	-	-
DOX-NP	35.64	41.98	50.38	44.55	13.28	13.42	-	-	-	-
BL-NP -BS	40.84	48.63	34.28	30.65	18.48	18.87	2.47	1.54	0.07	0.03
DOX- BS-NP	34.29	42.41	48.84	45.35	10.37	11.00	1.24	0.80	0.08	0.04

Note: Weight % and atomic % of elements in various nanoparticles.

Abbreviations: CK, carbon counts; OK, oxygen counts; NK, nitrogen counts; NaK, sodium count; SK, sufur count; BL-NP, blank nanoparticles; DOX-NP, doxorubicin loaded PLGA nanoparticles; BL-NP-BS, blank nanoparticles cross-linked with BS3; DOX-BS-NP, doxorubicin-loaded PLGA nanoparticles cross-linked with BS3.

Figure 2 In-vitro drug release profile of DOX from DOX-NP in different buffers (citrate buffer (pH 3), acetate buffer (pH 5), phosphate buffer (pH 7.4), and bicarbonate buffer (pH 10)) and different media (PBS release media added with hydroxy propyl β-cyclodextrin (HBC)/TWEEN, tween 80). Data show mean±SD (n=3).

Abbreviations: PVA, poly (vinyl alcohol); TPGS, tocopherol polyethylene glycol succinate; DOX, doxorubicin; Ab, antibody; NP, nanoparticle.

Table 3 In-vitro drug release kinetic data tested on various release kinetic models with corresponding R² values and release exponent (n) (Korsmeyer–Peppas model)

	Phosphate- buffered saline pH 7.4	HBC	Tween	Acetate buffer pH 5	Citrate buffer pH 3	Bicarbonate buffer pH 10
Zero order	y=0.0077x +4.0662	y=0.0102x +5.7819	y=0.0141x +11.237	y=0.0141x +30.183	y=10.308x +40.221	y=0.0052x +3.1674
R^2=0.9329	R^2=0.92	R^2=0.8128	R^2=0.6313	R^2=0.8679	R^2=0.9068
First order	y=−4E-05x +1.9826	y=−6E-05x +1.9751	y=−9E-05x +1.9475	y=−0.0001x +1.8338	y=−0.2231x +1.929	y=−2E-05x +1.9862
R^2=0.9504	R^2=0.9436	R^2=0.8463	R^2=0.7145	R^2=0.9682	R^2=0.9209
Higuchi	y=0.464x +0.1319	y=0.6156x +0.5097	y=0.8914x +2.9913	y=0.9629x +20.23	y=34.366x +16.017	y=0.3156x +0.4174
R^2=0.9948	R^2=0.9915	R^2=0.9565	R^2=0.8611	R^2=0.9446	R^2=0.9958
Hixon–Crowell	y=−0.0001x +4.5791	y=−0.0002x +4.5525	y=−0.0003x +4.4589	y=−0.0003x +4.0965	y=−0.4348x +4.104	y=−9E-05x +4.5924
R^2=0.9449	R^2=0.9362	R^2=0.8358	R^2=0.688	R^2=0.9765	R^2=0.9163
Korsmeyer–Peppas	y=0.406x− 0.0626	y=0.3856x +0.1296	y=0.385x +0.3448	y=0.2291x +1.0555	y=0.3072x +1.7477	y=0.4304x− 0.2729
	R^2=0.9735	R^2=0.9634	R^2=0.9776	R^2=0.9833	R^2=0.9921	R^2=0.9841
	n=0.4	n=0.38	n=0.38	n=0.22	n=0.3	n=0.43

Figure 3 Different studies for confirmation of antibody conjugation on the surface of polylactic-co-glycolic acid (PLGA) nanoparticles.

Notes: (A) Flow cytometry data for unconjugated nanoparticles, (B) for antibody-conjugated nanoparticles, (C) laser-scanning confocal microscopy, showing FITC labeled CD-340 antibody-conjugated scattered nanoparticles and (D) SDS-PAGE gel electrophoresis, showing CD-340 antibody-conjugated NP,  antibody unconjugated NP and free-antibody.

Abbreviations: DOX, doxorubicin; Ab, antibody; NP, nanoparticle.

Figure 4 In-vitro cytotoxicity data of DOX, DOX-NP, and DOX-Ab-NP determined in SKBR-3 cells, MCF-7 cells, and MDA-MB-231 cells. Data show mean ±SD (n=3).

Abbreviations: DOX, doxorubicin; Ab, antibody; NP, nanoparticle.

Figure 5 Cellular uptake of antibody conjugated and unconjugated nanoparticles in various cell lines.

Notes: (A) Cellular uptake of DOX-NP and DOX-Ab-NP in SKBR-3, MCF-7, and MDA-MB-231 cells observed by confocal microscopy at 1 and 6 hrs, respectively (B) Cellular uptake of antibody-conjugated blank nanoparticles in SKBR-3, MCF-7, and MDA-MB-231 cells observed by confocal microscopy at 6 hrs.

Abbreviations: DOX, doxorubicin; Ab, antibody; NP, nanoparticle.

Figure 6 Apoptosis and cell cycle analysis data of breast cancer cells treated with experimental formulations and free-drug.

Notes: (A) Apoptosis study by flow cytometry of SKBR-3 cells with the treatment of free DOX, DOX –NP, and DOX-Ab-NP after 24 hrs. Cell cycle analysis by flow cytometry for (B) SKBR-3 cells and (C) MCF-7 cells treated with DOX, DOX-NP, and DOX-Ab-NP for 24 hrs.

Abbreviations: DOX, doxorubicin; Ab, antibody; NP, nanoparticle.

Figure 7 Comparative HER2 levels, caspase activity, and PARP cleavages in SKBR-3 cells, MCF-7 cells, and MDA-MB-231 cells.

Notes: (A) Determination of the relative level of HER2 in SKBR-3 cells, MCF-7 cells, and MDA-MB-231 cells. (B) Activation of Caspase in MDA-MB-231 cells, MCF-7 cells, and SKBR-3 cells. (C) Detection of PARP cleavage in MDA-MB-231 cells, MCF-7 cells, and SKBR-3 cells. The values of p show statistical level of significance, **p<0.05, ***p<0.01, comparison between the groups are shown by over-head lines.

Abbreviations: DOX, doxorubicin; Ab, antibody; NP, nanoparticle.

Table 4 Plasma pharmacokinetic parameters of free doxorubicin and doxorubicin released from DOX-NP and DOX-Ab-NP after intravenous bolus administration of DOX, DOX-NP, and DOX-Ab-NP with an equivalent amount of drug in BALB/c mice

Parameters	Plasma values of drug
Upon DOX administration	Upon DOX-NP administration	Upon DOX-Ab-NP administration
t1/2 (hrs)	17.86±2.1	27.02±2.5^a	32.78±2.9^a
Tmax (hrs)	0.25±0.021	0.25±0.02	0.25±0.023
Cmax (ng/mL)	910.9±50.5	737.1±44.3^a	558.5±37.9^a,b
^§AUClast (hrs.ng/mL)	2880.1±199.2	3702.2±349.3	5663.5±440.2^a,b
AUCinf (hrs.ng/mL)	2934.2±205.32	3986.8±333.33^a	6382.5±448.4^a,b
AUMClast (hrs.hrs.ng/mL)	46,707.8±2202.6	81,206.6±7111.5^a	150,243.2±8200.1^a,b
AUMCinf (hrs.hrs.ng/mL)	53,296.5±2600	119,611.4±1000.2^a	253,262.5±1665.5^a,b
MRTlast (hrs)	16.2±0.99	21.9±1.5^a	26.5±2.02^a,b
MRTinf (hrs)	18.2±0.86	30.0±2.1^a	39.7±2.33^a,b
Vss (L/Kg)	61.9±4.8	75.2±4.99^a	62.2±5.2 ^b
CL (L/hrs/kg)	3.4±0.24	2.5±0.19^a	1.6±0.11 ^a,b

Notes: Significant difference corresponding from (I) DOX treatment ^ap<0.05, (ii) DOX-NP treatment ^bp<0.05. ‡Data show mean ± SD (n=3). ^§Units of AUC in plasma are hrs ng/mL, and in hepatic tissue hrs ng/g, respectively.

Abbreviations: DOX, doxorubicin; NP, nanoparticle; Ab, antibody; DOX, Free-drug, doxorubicin; DOX-NP, doxorubicin-loaded nanoparticles; DOX-Ab-NP, antibody conjugated doxorubicin loaded nanoparticles; t_1/2, half-life; AUC _last, area under the plasma concentration–time curve from time 0 to time of last measurable concentration; AUC_inf, under the plasma concentration–time curve from time 0 infinity; AUMC_last, area under the first moment curve from time 0 to time of last measurable concentration; AUMC_inf, area under the first moment curve from time 0 to infinity; MRT, mean resident time; V_ss, steady state volume of distribution; CL, clearance.

Figure 8 Plasma drug concentration upon IV bolus injection (at a dose of 10 mg/kg body weight) of DOX, DOX-NP, and DOX-Ab-NP. The same curve (time point 12–96 hrs) has been shown in the magnified version in the inset. Data show mean± SD (n=3).

Abbreviations: DOX, doxorubicin; Ab, antibody; NP, nanoparticle.

Table 5 Doxorubicin-induced cardiotoxicity parameters of different formulations after 2 weeks of final dosing

Treatment group	Serum CK-MB (U/L)*	LDH (IU/L)*	% SOD*	Heart weight (g)*
Control (without any drug treatment)	44.44±3.1	142.32±8.12	100±7.3	0.1722±0.03
BL-NP	55.54±4.4	149.0±8.5	98.65±7.0	0.1662±0.022
Free-DOX	126.65±7.0^a,b	250.10±11.7^a,b	34.02±4.5^a,b	0.1420±0.025
DOX-NP	70.20±5.6^a,b,c	180.39±9.23^a,b,c	42.78±5.1^a,b	0.1650±0.019
DOX-Ab-NP	66.63±6.1^a,b,c	166.67±8.8^c	49.22±5.5^a,b	0. 1670±0.036

Notes: *Data show mean ± SD (n=3). Significant difference corresponding from control ^ap<0.05, BL-NP; ^bp<0.05, Free-DOX; ^cp<0.05.

Abbreviations: CK-MB, creatine phosphokinase; LDH, lactate dehydrogenase; SOD, superoxide dismutase; BL-NP, blank nanoparticles; DOX, doxorubicin; DOX-NP, doxorubicin-loaded nanoparticles; DOX-Ab-NP, doxorubicin loaded antibody-conjugated nanoparticles.

Figure 9 Comparative cardiotoxicity profiles by the treatment of DOX, BL-NP, DOX-NP, and DOX-Ab-NP.

Notes: (A) Echocardiography images. (B) Histopathology of cardiac tissue (Vascularization due to free-drug treatment was shown by the green arrow in the respective image).

Abbreviations: DOX, doxorubicin; Ab, antibody; NP, nanoparticle.

Figure 10 Biodistribution of drug/formulation data and, tumor and tumor-related data in experimental animals.

Notes: (A) Biodistribution and accumulation data of ^99mTc labeled drug/formulations in experimental mice (a) time-dependent biodistribution and accumulation of ^99mTc-DOX at 0.5 hrs (d) and at 4 hrs, (b) ^99mTc -DOX-NP at 0.5 hrs (e) and 4 hrs, and (c) ^99mTc -DOX-Ab-NP at 0.5 hrs and (f) 4 hrs, in xenograft mice-bearing breast tumor. (B) Changes in tumor volume, animal body weight, and excised tumors from the experimental mice along with the tumor growth-related representative visual image data. (a) Relative changes in tumor volume with time. Data showed mean tumor volume (mean ± SD, n=3) in different experimental and control mice. The value p<0.05 shows the level of statistical significance. Prefix of p (i.e., a–j), indicates as mentioned below: “a” indicates statistical level of significance when data were compared against normal saline treated (control) mice on day 10; “b” indicates statistical level of significance when data were compared against DOX-treated mice on day 10; “c” indicates statistical level of significance when data were compared against normal saline treated (control) mice on day 15; “d” indicates statistical level of significance when data were compared against DOX-treated mice on day 15; “e” indicates statistical level of significance when data were compared against normal saline treated (control) mice on day 20; “f” indicates statistical level of significance when data were compared against DOX-treated mice on day 20; “g” indicates statistical level of significance when data were compared against DOX-NP-treated mice on day 20; “h” indicates statistical level of significance when data were compared against normal saline treated (control) mice on day 25; “i” indicates statistical level of significance when data were compared against DOX-treated mice on day 25; “j” indicates statistical level of significance when data were compared against DOX-NP-treated mice on day 25. (b) Change of body weight of nude mice-bearing tumor. Data represent mean ± SD (n=3); data showed that there was no statistically significant variation between the groups of mice. (c) Tumors excised from the experimental mice after treatment with saline/DOX/DOX-NP/DOX-Ab-NP. (d) Images on day 25 of nude mice bearing SKBR-3 tumor after treatment with saline/DOX/DOX-NP/DOX-Ab-NP. Tumor areas have been marked with red circles in the figure.

Abbreviations: ^99mTc-DOX: radiolabeled free-doxorubicin, ^99mTc-DOX -NP: radiolabeled doxorubicin loaded nanoparticles, ^99mTc-DOX-Ab-NP: radiolabeled doxorubicin loaded antibody conjugated nanoparticles; DOX, doxorubicin; Ab, antibody; NP, nanoparticle.

Table 6 Biodistribution data of ^99mTc-DOX, ^99mTc-DOX-NP, ^99mTc-DOX-Ab-NP in mice after IV. administration

Organ/Tissue	^99mTc-DOX	^99mTc-DOX-NP	^99mTc-DOX-Ab-NP
Blood	0.118±0.04	0.223±0.031^a	0.526±0.05^a,b
Heart	0.125±0.035	0.065±0.008^a	0.006±0.001^a,b
Liver	5.854±0.13	17.67±2.7^a	18.231±3^a
Lung	0.153±0.044	2.561±0.18^a	2.926±0.19^a
Spleen	0.052±0.01	0.122±0.06	0.351±0.052^a,b
Muscle	0.042±0.012	0.039±0.01	0.028±0.009
Kidney	5.380±0.18	2.181±0.11^a	2.921±0.21^a,b
Intestine	2.990±0.14	4.245±0.19^a	5.105±0.25^a,b
Stomach	0.167±0.05	0.223±0.03	0.251±0.04
Urine	21.25±3.6	12.214±2.6^a	11.91±2.1^a
Tumor	0.100±0.045	0.167±0.06	0.356±0.044^a,b

Notes: *Data show mean ± SD (n=3). Significant difference corresponding from DOX ^ap<0.05, DOX-NP; ^bp<0.05.

Abbreviations: DOX, doxorubicin; Ab, antibody; NP, nanoparticle; ^99mTc-DOX, radiolabeled free-doxorubicin; ^99mTc-DOX –NP, radiolabeled doxorubicin-loaded nanoparticles; ^99mTc-DOX-Ab-NP, radiolabeled doxorubicin-loaded antibody-conjugated nanoparticles.