X. Kuang et al.
Colloids and Surfaces B: Biointerfaces 203 (2021) 111766
animals were intravenously administered oxaliplatin solution, Oxa-OA
NPs and Oxa-SS-OA at 3 mg Pt/kg (n = 5). At predetermined time
points (15 min, 1, 4, 8, 12 h), blood samples were collected and
extracted to obtain the plasma. Nitric acid (HNO3, 65 %, supra pure) was
added into the plasma and allowed them to digest overnight. Then the
samples were heated to remove HNO3. Then Pt concentration was
measured with ICP-MS.
filaments for 10 s until paw withdraw as a response. In presence of a
response, the filament of the next lower force was applied. And in the
absence of a response, the filament of the next greater force was applied.
To avoid injury during tests, the cutoff strength of the von Frey filament
was 60 g. Each trial was repeated 3 times at approximately 2-min in-
tervals. The mean value was used as the force produced a withdrawal
response and recorded. And the body weights of all groups were also
recorded every week.
2.13. In vivo biodistribution
2.16. Statistical analysis
The in vivo distribution behaviors of Pt(IV) NPs into tumor and other
organs were assessed in 4T1 tumor-bearing Balb/c mice (female, n = 3).
Mice with subcutaneous tumors of approximately 400 mm3 were sub-
jected to tail vein injection of oxaliplatin solution, Oxa-OA NPs and Oxa-
SS-OA at 6 mg Pt/kg. At 4 and 12 h after dosing, mice were sacrificed
followed by the organs (heart, liver, spleen, lung and kidney) and tumors
immediately harvested. Then organs and tumors were washed in the
saline, weighted and sheared by a tissue homogenizer. Pt concentration
in tissues and tumors were analyzed by ICP-MS.
All quantitative data are expressed as the mean ± SD. Statistical
analysis was performed with Student’s t-test and one-way ANOVA. (*
p < 0.05, ** p < 0.01, *** p < 0.001.).
3. Results and discussion
3.1. Synthesis of Pt(IV) prodrugs
Unsaturated fatty acids based drug delivery strategies, characterized
by favorable biocompatibility and native tumor-targeting effect, have
been prevalently applied for the rational design of prodrug chemo-
therapy [26,29]. Several prodrugs have already successfully had access
to clinical trials [26].
2.14. In vivo antitumor efficacy and systematic toxicity investigation
Maximum tolerated dose (MTD) was determined before antitumor
efficacy investigation was conducted. Briefly, a series of doses of
different formulations were i.v. injected into female Balb/c mice (n = 3
each group) every other day. Total four injections were carried out on
the premise of no systemic toxicity observed. Body weights of mice were
monitored and the maximal dosage administrated by mice group was
determined as MTD if there were no more than 15 % weight loss of every
In this research, a GSH-responsive oxaliplatin prodrug monomer,
Oxa-SS-OA, was synthesized by introducing a GSH-cleavable disulfide
functional moiety to covalently bond oleic acid and oxaliplatin. Syn-
thesis routes of Pt(IV) prodrug and chemical structures confirmed by MS
and 1H NMR were shown in Fig. 2A and Figure S1 and S2.
mouse. Then 4T1 cells with a density of 5 × 106 cells per 100
μL were
injected subcutaneously on female Balb/c mice. When tumor volume
reached approximately 150 mm3, 4T1 tumor-bearing Balb/c mice were
randomly divided into 4 groups (n = 6) and intravenously injected with
5% glucose solution as control group, oxaliplatin solution, Oxa-OA NPs
and Oxa-SS-OA NPs at 6 mg Pt/kg. The i.v. administration schedule
included total 4 times injection at 6-day intervals and the first injection
was designated as day 0. Tumor volume and body weight was measured
every other day. After tumor monitoring, all mice (6 mg Pt/kg) were
euthanized and their blood samples were collected for hepatorenal
function test (ALT, AST, BUN and CREA), tissues (heart, liver, spleen,
lung, and kidney) separated for H&E staining. Tumors were harvested
and weighed after euthanasia.
3.2. Preparation and characterization of Pt(IV) self-assembled
nanoparticles
Both synthesized Oxa-OA and Oxa-SS-OA are able to self-assemble
into NPs in aqueous solution via nanoprecipitation. In contrast, free
oxaliplatin disperse in water uniformly suggesting the essential part of
oleic acid moiety in the nanoparticle-forming process. The size distri-
bution and morphology of nanoassemblies were characterized by TEM
and DLS in Fig. 2B–E and Figure S3. The average hydrodynamic diam-
eter of Oxa-OA and Oxa-SS-OA NPs was around 90 nm and 100 nm. And
the Zeta potential of both Pt(IV) nanoassemblies was ꢀ 20 mV.
(Table S1). The representative TEM images showed that both Pt(IV) NPs
displayed uniform spherical morphology with a particle size which is
comparable to that established by DLS measurement. Both Oxa-OA and
Oxa-SS-OA could self-assemble into NPs in absence of surfactant.
Without the aid of DSPE-PEG2000 in the assembly process, increasing size
and PDI were observed as shown in Table S1. Given possible undesired
stability [27] and in vivo drug behavior [30] of non-PEGylated formu-
lations, PEGylated Pt(IV) NPs (described as Oxa-OA NPs and Oxa-SS-OA
NPs) were applied for subsequent experiments.
2.15. Behavioral assessment of mechanical hypersensitivity
The behavior investigation was conducted to evaluate oxaliplatin-
induced peripheral neuropathy in SD rats subject to administration of
oxaliplatin solution, Oxa-OA NPs and Oxa-SS-OA NPs. An in vivo model
for oxaliplatin-induced peripheral neurotoxicity (increased sensitivity to
mechanical force) was used [28] and neurotoxicity that developed after
days following serial intravenous (i.v.) injections of oxaliplatin solution
were recorded.
Sensitivity baseline of normal rats to mechanical stimuli was estab-
lished by measuring 3 times before administration. Rats were randomly
assigned to 4 treatment groups (5% glucose, oxaliplatin solution, Oxa-
OA NPs and Oxa-SS-OA NPs, n = 10). Repeated dosing of 5% glucose
and different formulations at the same dose of 2 mg Pt/kg (twice a week
for a total of 4 i.v. injections) was launched to induce neuropathy.
Sensitivity thresholds of each animal measurement was performed
before drug administration.
3.3. Colloidal stability
The linkages impose great impact on the assembly manners of pro-
drug and colloidal stability of nanoparticles. Disulfide bonds between
cysteine residues make an essential contribution to the structural sta-
bility of proteins [31]. The space flexibility and self-assembly capacity
could be greatly improved by the existence of the bond angle or dihedral
angle of disulfide bonds which contribute to the stability of proteins or
nanoassemblies [21,32]. As shown in Figure S4, the PEGylated prodrug
NPs showed impressive colloidal stability with negligible size changes in
10 mM PBS containing 10 % FBS for 12 h. Besides, both Oxa-OA and
Oxa-SS-OA NPs remained stable in 5% glucose and RPMI 1640 medium
after being stored for seven days at room temperature. (Figure S5)
Specifically, rats were placed in individual transparent boxes and get
accustomed for 30 min. Mechanical paw withdrawal threshold (PWT) of
hindpaw in response to the vertical force of von Frey filaments was
recorded. A series of calibrated von Frey filaments (eleven levels ranging
from 0.6ꢀ 60 g, U.S. North Coast NC12775ꢀ 99) were applied perpen-
dicularly to the plantar surface of the hindpaw with a force to bend the
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