Lumbrokinase/paclitaxel nanoparticle complex: potential therapeutic applications in bladder cancer

Figures & data

Table 1 The stirring and dialyzing time and MWCO of the copolymers synthesis

Reaction mixture	Stirring (day)	Dialyzing (day)	MWCO (kDa)
PEG-b-PBLG	3	3	7
PEG-b-PELG	2	3	3.5
PEG-b-(PELG-g-PZLL)	3	3	7
PEG-b-(PELG-g-(PZLL-r-PLL))	3	3	7

Abbreviations: MWCO, molecular weight cutoff; PEG-b-PBLG, poly(ethylene glycol)-b-poly(γ-benzyl l-glutamate); PEG-b-PELG, poly(ethylene glycol)-b-poly (ethylenediamine l-glutamate); PEG-b-(PELG-g-PZLL), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)).

Table 2 MW, TEM, LK-, and PTX-loading capacity of the nanocarrier

Sample	Mn (kDa)/^1H NMR	TEM (nm)	Loading capacity (%)
P	34.5	41±2	NA
LK/P	NA	83±3	6.74
PTX/P	NA	78±2	4.13
LK/PTX/P	NA	89±3	10.34a

Notes:

a The negatively charged LK combines with a positively charged PLL via electrostatic interactions and hydrophobic PTX combines with PZLL through hydrophobic interaction at pH 7.4, respectively.

Abbreviations: ¹H NMR, ¹H nuclear magnetic resonance; LK, lumbrokinase; MW, molecular weight; NA, not applicable; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PLL, poly(l-lysine); PZLL, poly(ε-benzyoxycarbonyl-l-lysine); PTX, paclitaxel; TEM, transmission electron microscopy.

Figure 1 Synthesis of PEG-b-(PELG-g-(PZLL-r-PLL)).

Note: x, y, m, and n represent the degree of polymerization.

Abbreviations: 2-HP, 2-hydroxypyridine; DMF, N,N-dimethylformamide; PEG-NH₂, poly(ethylene glycol) amine; PEG-b-PBLG, poly(ethylene glycol)-b-poly(γ-benzyl l-glutamate); PEG-b-PELG, poly(ethylene glycol)-b-poly(ethylenediamine l-glutamate); PEG-b-(PELG-g-PZLL), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly (ε-benzyoxycarbonyl-l-lysine)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)).

Figure 2 Characterization of nanocomplexes.

Notes: (A) ¹H NMR spectra of PEG-b-(PELG-g-(PZLL-r-PLL)); (B) TEM image of copolymers, (a) PEG-b-(PELG-g-(PZLL-r-PLL)), (b) LK/PEG-b-(PELG-g-(PZLL-r-PLL)), (c) PTX/PEG-b-(PELG-g-(PZLL-r-PLL)), and (d) LK/PTX/PEG-b-(PELG-g-(PZLL-r-PLL)); (C) Loading pattern diagram of PEG-b-(PELG-g-(PZLL-r-PLL)); (D) Cumulative releasing profile of LK and PTX from PEG-b-(PELG-g-(PZLL-r-PLL)) complexes (n=5); (E) Relative cell viability (n=5); (F) Absorbance value of cell proliferation (n=5); (G) Inhibition ratio of cell proliferation (n=5). Data are expressed as the mean ± SD. *p<0.01 vs control, **p<0.05 vs LK or PTX, ***p<0.01 vs LK/P or PTX/P.

Abbreviations: ¹H NMR, ¹H nuclear magnetic resonance; LK, lumbrokinase; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)); PTX, paclitaxel.

Figure 3 Cell apoptosis.

Notes: (A) Control; (B) LK; (C) PTX; (D) LK/P; (E) PTX/P; (F) LK/PTX/P; (G) Cell apoptosis rate. Scale bar 40 µm. Data are expressed as the mean ± SD, n=5. *p<0.01 vs Control, **p<0.05 vs LK or PTX, ***p<0.01 vs LK/P or PTX/P.

Abbreviations: LK, lumbrokinase; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)); PTX, paclitaxel.

Figure 4 Blood concentration of LK and PTX.

Notes: (A) Blood LK concentration; (B) Blood PTX concentration. Data are expressed as the mean ± SD, n=7.

Abbreviations: LK, lumbrokinase; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)); PTX, paclitaxel.

Figure 5 Lifespan and life prolongation rate.

Notes: (A) Lifespan; (B) Life prolongation rate. Data are expressed as the mean ± SD, n=10. *p<0.01 vs Control, **p<0.05 vs LK or PTX, ***p<0.01 vs LK/P or PTX/P.

Abbreviations: LK, lumbrokinase; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)); PTX, paclitaxel.

Figure 6 Average tumor weight and tumor inhibition rate.

Notes: (A) Average tumor weight; (B) Tumor inhibition rate. Data are expressed as the mean ± SD, n=10. *p<0.01 vs Control, **p<0.05 vs LK or PTX, ***p<0.01 vs LK/P or PTX/P.

Abbreviations: LK, lumbrokinase; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)); PTX, paclitaxel.

Table 3 The bladder cancer incidence in rats

Group	Transitional-cell carcinoma		Canceration rate (%)
	Class I and <Class I	>Class I
Control	11/20	6/20	85.0
LK	6/20	5/20	55.0
PTX	5/20	5/20	50.0
LK/P	4/20	3/20	35.0
PTX/P	4/20	2/20	30.0
LK/PTX/P	3/20	2/20	25.0

Abbreviations: LK, lumbrokinase; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PTX, paclitaxel.

Figure 7 Histopathology.

Notes: (A) Control; (B) LK; (C) PTX; (D) LK/P; (E) PTX/P; (F) LK/PTX/P. Disorganized cells, pathological mitotic figure, and papillary hyperplasia are observed in local or multiple regions in the Control group; the cell abnormality in LK and PTX groups is lower than that of the Control group; the cell abnormality in the LK/P and PTX/P groups is lower than that of the LK and PTX groups; and the cell abnormality in the LK/PTX/P group is significantly lower than that of the LK/P and PTX/P groups. Hematoxylin-Eosin staining, magnification ×100.

Abbreviations: LK, lumbrokinase; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)); PTX, paclitaxel.

Figure 8 CD34 expression and MVD in the rat bladder cancer tissue.

Notes: (A) Control; (B) LK; (C) PTX; (D) LK/P; (E) PTX/P; (F) LK/PTX/P; (G) MVD. Immunohistochemical staining, magnification ×200. Data are expressed as the mean ± SD, n=10. *p<0.01 vs Control, **p<0.05 vs LK or PTX, ***p<0.01 vs LK/P or PTX/P.

Abbreviations: HPF, high-power field in an optical microscope; LK, lumbrokinase; MVD, microvessel density; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-Lysine)); PTX, paclitaxel.

Figure 9 Expression of p53 and cyclin B1 in the rat bladder cancer tissue.

Notes: Data are expressed as the mean ± SD, n=10. *p<0.01 vs Control, **p<0.05 vs LK or PTX, ***p<0.01 vs LK/P or PTX/P.

Abbreviations: LK, lumbrokinase; P, PEG-b-(PELG-g-(PZLL-r-PLL)); PEG-b-(PELG-g-(PZLL-r-PLL)), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly (ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)); PTX, paclitaxel.