Targeting delivery of Radix Ophiopogonis polysaccharide to ischemic/reperfused rat myocardium by long-circulating macromolecular and liposomal carriers

Figures & data

Table 1 Characterization of ROP and the conjugates by HPGPC

Samples	RT (min)	Mn (Da)	Mw (Da)	Mp (Da)	Polydispersity index	Grafting degree
ROP	18.63	3,004.7	3,622.8	3,136.4	1.20	–
PEG20k-NH2	15.34	24,691.6	25,709.9	25,564.0	1.04	–
P20k-R	15.21	26,471.1	27,670.5	27,628.81	1.04	0.960
PEG40k-NH2	14.57	34,873	41,380	48,015	1.19	–
P40k-R	14.44	35,724	42,721	49,479	1.20	0.983

Abbreviations: ROP, Radix Ophiopogonis polysaccharide; HPGPC, high-performance gel permeation chromatography; RT, residence time; min, minutes; PEG, polyethylene glycol; P_20k-R, 20-kDa PEG mono-modified ROP; P_40k-R, 40-kDa PEG mono-modified ROP; Mn, number average molecular weight; Mw, weight average molecular weight; Mp, peak average molecular weight.

Table 2 Pharmacokinetic parameters of ^FR, ^FP_20k-R, ^FP_40k-R, and ^FR loaded in L-Lp after intravenous injection at a same molar dose of 4 μmol/kg in rats (n=3)

Parameters	^FR	^FP20k-R	^FP40k-R	^FR loaded in L-Lp
AUC(0–t) (h μmol/L)	4.81±0.39	244.01±9.03**	549.66±31.87**,##	555.03±62.27**,##
AUC(0–∞) (h μmol/L)	5.82±0.37	251.71±8.86**	660.46±63.34**,##	629.79±66.56**,##
MRT(0–∞) (h)	0.33±0.02	8.03±0.30**	25.09±3.00**,##	20.49±0.31**,##
t1/2 (h)	0.39±0.03	12.67±0.20**	20.51±2.58**,#	17.65±1.14**,#
V (L/kg)	0.23±0.03	0.128±0.008*	0.152±0.004*,##	0.131±0.016**
CL (mL/h/kg)	6.90±0.40	15.91±0.55**	6.09±0.58**,##	6.40±0.69**,##

Notes:

** P<0.01, compared with rats in the ^FR group;

* P<0.05, compared with rats in the ^FR group;

## P<0.01, compared with rats in the ^FP_20k-R group;

# P<0.05, compared with rats in the ^FP_20k-R group. Data are presented as mean ± standard deviation.

Abbreviations: ^FR, FITC-labeled ROP; ^FP_20k-R, FITC-labeled 20-kDa PEG mono-modified ROP; ^FP_40k-R, FITC-labeled 40-kDa PEG mono-modified ROP; L-Lp, long-circulating liposomes; AUC, area under the curve; h, hour; MRT, mean residence time; FITC, fluorescein isothiocyanate; PEG, polyethylene glycol; ROP, Radix Ophiopogonis polysaccharide; CL, systemic clearance.

Figure 1 Blood clearance of ^FR (■), ^FP_20k-R (□), ^FP_40k-R (○), and ^FR loaded in L-Lp (Δ) after intravenous injection at a same molar dose of 4 μmol/kg in rats (n=3).

Abbreviations: ^FR, FITC-labeled ROP; ^FP_20k-R, FITC-labeled 20-kDa PEG mono-modified ROP; ^FP_40k-R, FITC-labeled 40-kDa PEG mono-modified ROP; L-Lp, long-circulating liposomes; h, hour; FITC, fluorescein isothiocyanate; PEG, polyethylene glycol; ROP, Radix Ophiopogonis polysaccharide.

Table 3 The ratio of fluorescent intensity for each myocardial area in drug-treated or -untreated IR rats to that for myocardium in normal rats without drug treatment (n=3)

Drug	Infarcted area		Noninfarcted area
	3 h	12 h	3 h	12 h
No drug	1.04±0.12	1.04±0.07	1.05±0.11	0.99±0.09
^FP20k-R	10.48±0.39*	4.01±0.56*	3.54±0.08*	1.56±0.09*
^FP40k-R	6.63±1.67*	4.75±0.18*	2.45±0.46*	1.98±0.06*
^FR loaded in L-Lp	24.79±3.72*	23.34±7.61*	2.98±0.38*	2.74±0.47*

Note:

* P<0.01, compared with the group without drug treatment. Data are presented as mean ± standard deviation.

Abbreviations: IR, ischemia/reperfusion; h, hour; ^FP_20k-R, FITC-labeled 20-kDa PEG mono-modified ROP; ^FP_40k-R, FITC-labeled 40-kDa PEG mono-modified ROP; ^FR, FITC-labeled ROP; L-Lp, long-circulating liposomes; FITC, fluorescein isothiocyanate; PEG, polyethylene glycol; ROP, Radix Ophiopogonis polysaccharide.

Figure 2 Fluorescent images of left ventricular transverse slices of rats.

Notes: Representative fluorescent images of left ventricular transverse slices taken from normal rats (A), normal rats that received ^FP_20k-R and were sacrificed at 12 hours postdose (B), IR rats (C), and IR rats that received ^FP_20k-R and were sacrificed at 12 hours postdose (D).

Abbreviations: ^FP_20k-R, FITC-labeled 20-kDa PEG mono-modified ROP; IR, ischemia/reperfusion; FITC, fluorescein isothiocyanate; PEG, polyethylene glycol; ROP, Radix Ophiopogonis polysaccharide.

Figure 3 Fluorescent images and TTC staining photos of left ventricular transverse slices of rats.

Notes: Representative fluorescent images (up) and the corresponding TTC staining photos (down) of left ventricular transverse slices taken from IR rats that received ^FP_40k-R (left) or L-Lp loaded with ^FR (right) and were sacrificed at 12 hours postdose.

Abbreviations: TTC, triphenyltetrazolium chloride; IR, ischemia/reperfusion; ^FP_40k-R, FITC-labeled 40-kDa PEG mono-modified ROP; L-Lp, long-circulating liposomes; ^FR, FITC-labeled ROP; FITC, fluorescein isothiocyanate; PEG, polyethylene glycol; ROP, Radix Ophiopogonis polysaccharide.

Figure 4 Changes in the fluorescent intensity of drugs with time postdose in infarcted myocardium (Is) and noninfarcted myocardium (N-Is) of IR rats.

Notes: For direct comparison among groups, the fluorescent intensities of ^FP_20k-R and ^FP_40k-R were converted to ones equivalent to ^FR via the molar fluorescent ratios of ^FP_20k-R and ^FP_40k-R to ^FR, which are 1.15 and 0.737, respectively. **P<0.01, *P<0.05, compared with Is at the same time point; ^##P<0.01, ^#P<0.05, compared with L-Lp in Is at the same time point; ^&&P<0.01, compared with ^FP_20k-R in Is at the same time point; ^$$P<0.01, compared with ^FP_20k-R in Is at 3 hours.

Abbreviations: IR, ischemia/reperfusion; ^FP_20k-R, FITC-labeled 20-kDa PEG mono-modified ROP; ^FP_40k-R, FITC-labeled 40-kDa PEG mono-modified ROP; ^FR, FITC-labeled ROP; L-Lp, long-circulating liposomes; min, minutes; h, hour; FITC, fluorescein isothiocyanate; PEG, polyethylene glycol; ROP, Radix Ophiopogonis polysaccharide.

Figure 5 The EPR effect-based infarcted myocardium targeting efficacy of ^FR, ^FP_20k-R, ^FP_40k-R, and L-Lp loaded with ^FR at different times postdose.

Notes: The targeting efficacy was calculated as the ratio of drug fluorescence intensity in the infarcted myocardium to that in the noninfarcted myocardium. ^##P<0.01, ^#P<0.05, compared with L-Lp at the same time point.

Abbreviations: EPR, enhanced permeability and retention; ^FR, FITC-labeled ROP; ^FP_20k-R, FITC-labeled 20-kDa PEG mono-modified ROP; ^FP_40k-R, FITC-labeled 40-kDa PEG mono-modified ROP; L-Lp, long-circulating liposomes; min, minutes; h, hour; FITC, fluorescein isothiocyanate; PEG, polyethylene glycol; ROP, Radix Ophiopogonis polysaccharide.