Angewandte
Chemie
Table 1: Antitumor potential of SN-38 prodrug-loaded PEG-PLA NPs
Interestingly, distinct release profiles were observed among
these NPs. NPs encapsulating prodrugs 8, 12, and 13 exhibited
slower release kinetics than 6- and 7-loaded NPs. To further
elucidate the correlation between the release and the
hydrolysis kinetics of prodrugs, we studied the hydrolysis of
7 and 8 as model compounds at neutral pH. Prodrug 8 was
found to be more kinetically inert to hydrolysis than 7 (see
Figure S3 in SI). This result is consistent with the release
profile for free SN-38 and indicates that this engineered
prodrug platform could deliver a sufficient quantity of active
agents to the target tumor sites by a simple hydrolysis process
that avoids the inefficient enzymatic activation required for
CPT-11. The prodrugs with enhanced lipophilicity might
readily bind to endogenous albumin, which would lead to the
destruction of the NPs. We thus verified the stability of 7- and
12-NPs in the presence of serum by DLS analysis. 7-loaded
NPs exhibited a partial peak shift in size distributions but still
remained stable within several hours. In contrast, 12-loaded
NPs were quite stable during the longtime incubation with
50% serum (see Figure S4 in SI), indicating that the drugs can
arrive at the tumor sites together with nanoparticles through
the EPR effect.
We next evaluated the cytotoxic effects of prodrug-loaded
NP candidates by measuring the half maximal inhibitory
concentration (IC50) of cell proliferation. Cell viability was
quantified using the standard MTT assay after 48 h of
treatment with prodrug-NPs. As shown in Figure 3a,b and
Table 1, these SN-38 prodrug-NPs exhibited high cytotoxicity
to all tested cancer cells and were approximately two orders
of magnitude more effective than CPT-11. Unexpectedly, the
antiproliferative activities induced by incubation with 8-, 12-,
and 13-NPs occurred at much lower concentrations than for
free SN-38 administered in DMSO in HCT-116, SW480, and
A549 cells (e.g., in the best case showing 26-fold potency).
The superior activity observed in vitro is impressive given that
the prodrug-NPs must undergo two steps to release active SN-
38 molecules as discussed above. This requirement in general
will result in reduced in vitro cytotoxicity in comparison to the
free drug. By contrast, the nonhydrolyzable ether-linked SN-
38 prodrug 1-NP exerted less cytotoxicity to all tested cancer
cells, with IC50 values greater than 3 mm.
To determine whether the inhibition of cancer cell
proliferation by these prodrug-NPs was a consequence of
SN-38-induced apoptosis, we conducted an Alexa Fluor 488
Annexin V/propidium iodide (PI) double-staining assay in
HCT-116 cells. The exposure of phosphatidylserine on the
outer leaflet of the cell membrane is an essential event in
apoptosis that can be specifically detected by the binding of
fluorescently labeled annexin V.[12] After exposure to CPT-11
(3 mm), free SN-38 (3 mm), and prodrug-NPs (3 mm, SN-38
equivalent doses) for 12 or 24 h, cells were analyzed by
fluorescence-activated cell sorting (FACS). Indeed, a high
level of apoptosis after 12 or 24 h of treatment was induced by
these prodrug-loaded NPs, which was comparable to the level
induced by free SN-38 (Figure 3c and Figure S5 in SI).
Together, these cell-based experiments clearly suggest that
these prodrug-loaded NPs can perform similarly to free SN-38
agents in vitro in effectively inducing apoptosis in cancer cells.
after 48 h of incubation (expressed as IC50 Æstandard deviation in mm).[a]
Cell line
HCT-116
SW480
A549
MCF-7
CPT-11
SN-38
21.6Æ1.8 28.6Æ2.5
20.4Æ2.0 88.5Æ20.8
0.22Æ0.05 0.26Æ0.06 0.31Æ0.02 1.43Æ0.22
6ꢁPEG8K-PLA16K 1.05Æ0.17 0.98Æ0.25 2.11Æ0.06 2.69Æ0.49
7ꢁPEG2K-PLA8K
2.19Æ0.34 1.63Æ0.20 4.53Æ0.08 2.27Æ0.61
8ꢁPEG5K-PLA16K 0.13Æ0.02 0.02Æ0.01 0.18Æ0.03 1.52Æ0.46
12ꢁPEG2K-PLA2K 0.13Æ0.05 0.04Æ0.02 0.19Æ0.02 1.21Æ0.27
13ꢁPEG5K-
0.11Æ0.06 0.01Æ0.004 0.17Æ0.03 1.36Æ0.31
PLA16K
[a] Determined by MTT assay.
To establish the clinical translation potential of prodrug-
encapsulated NPs, we performed therapeutic studies in vivo
using a HCT-116 colorectal xenograft model. To decrease the
number of required animals, only prodrug 6-, 7-, 8-, 12-, and
13-loaded NPs were tested and compared due to their high
in vitro activities and stability of formulation. Unfortunately,
the 13-formulated NP caused immediate death of mice during
intravenous injection; we thus terminated the in vivo ther-
apeutic procedure. The antitumor efficacy of 6-, 7-, 8-, and 12-
loaded NPs is illustrated in Figure 4a, c and d. The tumor
growth was remarkably inhibited after the successive intra-
venous injection of all prodrug-loaded NPs (at 10 mgkgÀ1 SN-
38 equivalent dose) as compared to saline and CPT-11
(12 mgkgÀ1) controls, thus demonstrating the superiority of
combining the drug reform strategy with nanoparticle-based
delivery platforms. In particular, the group treated with
prodrug 12-loaded NPs produced a more drastic decrease in
the tumor progression, resulting in a mean tumor volume of
215 mm3 versus 708 mm3 for CPT-11-treated control (n = 7,
p < 0.01). By comparison, untreated mice from the saline
group showed rapid tumor growth, with tumor volume
reaching approximately 1293 mm3 by day 20. The in vivo
distribution of drugs and consequently of their antitumor
efficacies rely heavily on factors such as the nanoparticle size,
surface characteristics, and shape.[13] Considering the similar
surface properties of the prodrug-encapsulated NPs (e.g.,
shapes and zeta potentials), we might partially attribute the
superior outcome of 12-loaded NPs to their higher cytotox-
icity in vitro and relatively small particle size (approximately
20 nm), which exceeds the 5 nm cutoff for clearance by the
kidney but may exhibit preferential accumulation in the
tumor site.[14] It was also notable that the in vivo efficacy of
the prodrug-formulated NPs was closely correlated with their
in vitro cytotoxicity, highlighting the value of using this
parameter in designing more efficient chemotherapeutics in
future work.
The body weights of the mice receiving treatment with
NPs all remained stable, suggesting a low systemic toxicity of
these prodrugs and related delivery materials (Figure 4b).
Although animal experiments with higher doses of prodrug-
loaded NPs were not conducted, elevated doses should be
expected to improve cancer therapy.[15]
In summary, as a proof of principle, we presented a drug
reform strategy for constructing a small library of lipophilic
SN-38 derivatives and screened their ability to be incorpo-
rated into amphiphilic NP formulations. Compared to the
Angew. Chem. Int. Ed. 2014, 53, 11532 –11537
ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wang, Hangxiang
