Supplementary material

A precision-guided MWNT mediated reawakening

the sunk synergy in RAS for anti-angiogenesis lung

cancer therapy

Yujie Su 1, †, Yahui Hu 1, †, Yu Wang 1, ‡, Xiangting Xu †, Yang Yuan †, Yunman Li *, †,

Zeyuan Wang §, Kerong Chen †, Fangrong Zhang †, Xuefang Ding †, Min Li †,

Jianping Zhou *, †, Yuan Liu †, Wei Wang *, †

† State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China

Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China

‡ Collaborative Innovation Center for Cardiovascular Disease Translational Medicine,

Department of Pharmacology, Nanjing Medical University, 140 Hanzhong Road,

Nanjing 210029, China

§ Department of Pharmaceutical Sciences, Temple University school of pharmacy,

3307 North Broad Street, Philadelphia , Pennsylvania 19140, USA

Author information

1 These authors contributed equally to this work

* Corresponding Authors

E-mail: wangcpu@163.com (W. Wang), zhoujp60@163.com (J. Zhou),

yucaoren@sina.com (Y. Li).

Notes

The authors declare no competing financial interest.

The stability and homogeneity study of f-MWNT and f-MWNT/pAT2 in mice

serum

The homogeneity and stability of f-MWNT and the corresponding

f-MWNT/pAT2 in biological media were represented as particle size and PDI changes

over time (Fig. S1). After f-MWNT and f-MWNT/pAT2 dispersed in mice serum for

48 h, both of them showed no significant changes in particle size with no more than

25 nm increase. Besides, the changes of PDI of f-MWNT and f-MWNT/pAT2 were

within 0.1 during the experiment. These results indicated our constructed

nanoparticles possessed favourable homogeneity and stability in biological media and

were feasible for in vivo drug delivery.

Figure S1. Serum stability of f-MWNT and f-MWNT/pAT2. (A) Particle size and PDI changes of

f-MWNT against time in mice serum. (B) Particle size and PDI changes of f-MWNT/pAT2 against

time in mice serum. Results were expressed as mean ± S.D. (n = 3).

The colloidal stability study of MWNT-COOH and

iRGD-PEI-MWNT-SS-CD/pAT2 nanocomplexes

The colloidal stability of MWNT-COOH and the final MWNT-based

nanocomplexes was presented as concentration changes of dispersion versus time

(Fig. 6). MWNT-COOH and iRGD-PEI-MWNT-SS-CD/pAT2 showed similar

tendency in concentration changes and no significant decrease of concentration in

distilled water after 14 days standing. However, MWNT-COOH precipitated

obviously in PBS buffer with concentration decreasing to 61.03% in day 14

comparing to that in the first day. iRGD-PEI-MWNT-SS-CD/pAT2 exhibited

preferable colloidal stability with concentration decreasing to 83.92% in day 14

comparing to that in the first day. It was supposed that functionalization of MWNT

with PEI and iRGD, which were hydrophilic molecules, improved the hydrophilicity

of the complexes and stabilized the dispersion system. Besides, PEI with high cationic

charge density effectively kept appropriate zeta potential of the complexes in

electrolyte solution, which also enhanced the colloidal stability of the complexes.

These results demonstrated our constructed iRGD-PEI-MWNT-SS-CD/pAT2

possessed superior colloidal stability.

Figure S2. The colloidal stability of MWNT-COOH and iRGD-PEI-MWNT-SS-CD/pAT2. (A)

Concentration changes of MWNT-COOH and iRGD-PEI-MWNT-SS-CD/pAT2 in distilled water

during 14 days. (B) Concentration changes of MWNT-COOH and

iRGD-PEI-MWNT-SS-CD/pAT2 in PBS buffer during 14 days. Results were expressed as mean ±

S.D. (n = 3).

Determination of grafting rate in f-MWNT

The contents of modifiers in f-MWNT were determined using TGA analysis in a

temperature change from 30℃ to 700℃ (Fig. S3A). The difference in the weight loss

at 400℃ between PEI-MWNT and MWNT-COOH implied that the PEI grafting

amount in PEI-MWNT was about 41.61 wt.%. For PEI-MWNT-SS-CD, its weight

loss comparing to MWNT-COOH revealed that total contents of PEI and SS-CD were

about 39.73 wt.%. Additional reference to the 1H-NMR of PEI-MWNT-SS-CD, paeks

area of PEI and SS (δ 2.30-3.15 ppm) and paeks area of CD (δ 1.21-1.44 ppm) figured

out the proportion between PEI and SS-CD were 1:2.28 in molar ratio, further

deduced their grafting amounts were about 22.98 wt.% and 16.75 wt.%, respectively.

For determining the iRGD contents, the weight loss of iRGD-PEI-MWNT and iRGD-

PEI-MWNT-SS-CD were comparing to corresponding f-MWNT without iRGD, and

iRGD grafting amounts in iRGD-PEI-MWNT and iRGD-PEI-MWNT-SS-CD were

14.76 wt.% and 10.11 wt.%, respectively.

Ellmen’s colorimetry was performed to reconfirm monosubstituted SS-CD

contents by survey of disulfide bond. PEI-MWNT-SS-CD processed with 5,5’-

dithiobis-2-nitrobenzoic acid (DTNB) was measured using UV spectra (Fig. S3B). By

calculating with standard curve which graphed using a series of SS of different

concentrations, the absorbance at 412 nm indicated the amount of monosubstituted

SS-CD in PEI-MWNT-SS-CD was 18.90 wt.% which was similar with TGA analysis.

Figure S3. Determination of grafting rate of each component on MWNT. (A) TGA analysis of

MWNT, MWNT-COOH, PEI-MWNT, iRGD-PEI-MWNT, PEI-MWNT-SS-CD and iRGD-PEI-

MWNT-SS-CD. (B) UV spectrum of PEI-MWNT-SS-CD with or without Ellmen’s method

treatment.

X-ray photoelectron spectroscopy analysis (XPS)

The spectra in XPS analysis revealed the elemental composition of the samples

(Tab. 1). In Pristine MWNT, the present of carbon accounted for 99.16 mol% with

minor oxygen of 0.84 mol%. However, the amounts of oxygen in MWNT-COOH

significantly increased to 11.18 mol% indicating successful introduction of oxygen-

containing groups. For PEI-MWNT and PEI-MWNT-SS-CD, the present of nitrogen

and sulfur respectively revealed the introduction of PEI and SS-CD. The increased

oxygen in PEI-MWNT-SS-CD towards PEI-MWNT might attribute to the oxygen in

CD. Although it was inaccuracy to assess the state of COOH residues and the

conjugation efficiency of the PEI molecules and drug due to the complexity of the

surface functional groups, these data reflected the chemical composition of the

samples to some extent.

Besides, typical C 1s spectra detailed the chemical state of carbon in samples

(Fig. 5). For Pristine MWNT, a single peak at 284.44 eV represented the carbon on

the sidewall. For MWNT-COOH, peaks at 283.5 eV and 283.9 eV stood for carbon on

the sidewall, C-H or C-OR bonds, peak at 288.02 eV corresponded to carboxyl

groups. For PEI-MWNT and PEI-MWNT-SS-CD, peaks at 286.9 eV and 286.4 eV

were due to emission from carbon atoms bound to one oxygen atom which were

presumably attributed to C=O bonds in the amide bonds formed between carboxyl

groups of MWNT and primary amine groups of PEI or SS-CD. Besides, peaks at

283.5 eV, 283.9 eV and 288.02 eV in MWNT-COOH respectively shifted to 283.79

eV, 284.9 and 290.2 eV in PEI-MWNT, and 283.7 eV, 284.8 eV and 288.9 eV in PEI-

MWNT-SS-CD. These shifts towards the higher energy also indicated the formation

of covalent bonds between MWNT and functional groups. Thus, the XPS spectra

confirmed the successful synthesis of f-MWNT.

Table S1. Elemental composition of the samples obtained by XPS analysis.

Samples C (mol%) N (mol%) O (mol%) S (mol%)

Pristine MWNT 99.16 - 0.84 -

MWNT-COOH 87.74 0.87 11.18 0.21

PEI-MWNT 80.14 12.82 7.04 -

PEI-MWNT-SS-CD 79.20 9.95 10.05 0.80

Figure S4. XPS spectra of pristine MWNT, MWNT-COOH, PEI-MWNT and PEI-MWNT-SS-CD.

(A) XPS full spectrum of pristine MWNT, MWNT-COOH, PEI-MWNT and PEI-MWNT-SS-CD.

(B) A typical C 1s spectrum of pristine MWNT, MWNT-COOH, PEI-MWNT and PEI-MWNT-

SS-CD.

pDNA release and DNase I protection assay

Successful gene therapy requires vectors to protect gene from the hostile in vivo

environment and release the cargo in the target site. pDNA entering systemic

circulation suffered from a rapid degradation by nuclease which presents as primary

obstacle [1, 2]. Fig. S5B showed no band was observed for DNase I treated naked

pDNA, indicating the vulnerability of naked pDNA in the presence of nuclease.

However gene in complexes at w/w ratio equal or greater than 6 were insusceptible

when facing to DNase I. The gene condensed by high mass of f-MWNT showed

strong resistance to nuclease with consistency in bands brightness comparing to

untreated pDNA. On the other hand, exposure of complexes to anionic heparin which

was simulated intracellular ion environment resulted clear bands of free pDNA on gel

even at a high w/w ratio of 16 (Fig. S5A), demonstrating that detachment of pDNA

from f-MWNT could be effectively achieved by ion-exchange manner to maximize

the transfection efficiency.

Figure S5. Characterization of the pDNA release ability and protecting effect to DNase I of f-

MWNT/pDNA complexes. (A) Gel retardation assay of f-MWNT/pDNA complexes at various

w/w ratios (f-MWNT : pDNA) in the present of polyanion heparin. (B) Gel retardation assay of f-

MWNT/pDNA complexes at various w/w ratios (f-MWNT : pDNA) in the present of DNase I.

In vitro release of CD

Drug carriers capable of fast releasing their payload at focused site tend to

produce improved therapeutic efficacy [3]. The disulfide bond which is dynamic and

reversible to oxidation and reduction conditions makes cystamine a redox-sensitive

switch of CD releasing from f-MWNT rather than a dull linkage. Hence CD release

behavior in vitro was performed in presence of different concentrations of GSH to

simulate reductive environment in vivo. Fig. S6 showed cumulative release rate of CD

from iRGD-PEI-MWNT-SS-CD/pDNA in the absent of GSH was only 8.5% of the

total CD in complexes at 48 h. Similar results of 10% and 9.4% were obtained in the

presence of mice serum and PBS with 2 μM GSH which mimicked the physiological

environment in blood circulation. Distinctively, when

iRGD-PEI-MWNT-SS-CD/pDNA exposed to intracellular level of GSH concentration

of 10 mM, CD release was remarkablely facilitated and reached 51.6% within 48 h.

Further increasing GSH concentration to 40 mM （ concentration in tumour

cells）resulted a dramatically accelerated release of CD reaching 78% at 48h. Above

data presenting in a GSH concentration dependence manner testified the function of

disulfide linkage in CD release control. The complexes were inferred to be highly

stable loading of CD after intravenously administering into circulation but smartly

unload cargo in tumor cells.

Figure S6. In vitro redox-triggered CD release from iRGD-PEI-MWNT-SS-CD/pDNA at 37 in℃

mice serum and in PBS in absence or presence of 2 μM, 10mM or 40mM GSH. The experiment

was performed in PBS (20 mM, pH 7.4) with iRGD-PEI-MWNT-SS-CD/pDNA concentration

fixed at 1.0 mg/mL. Data are presented as mean ± SD (n = 3).

Cytotoxicity assay

Nano-vectors for improving drug efficacy and reducing unintended side effects

are systemically administrated into circulation, these especially require carrier itself to

be safe and hypotoxic. The cytotoxicity of f-MWNT was evaluated by MTT assay on

A549 and HUVEC cell lines which were used in subsequent studies and contrastively

analyzed with PEI 25k, PEI 1.8k and MWNT-COOH (Fig. S7). PEI 25k, a common

positive transfection reagent, showed significant inhibitory effects on both cells

mainly due to its cationic nature and a noncleavable molecular structure [4]. The

cellular viabilities were about 57% for A549 and 66% for HUVEC after incubating

with PEI 25k at only 5 μg/mL for 24h and substantially reduced as PEI 25k

concentration increasing. On contrary, PEI 1.8k with a lower molecular weight

presented negligible influence on cellular viabilities. Four kinds of f-MWNT were

proved to have admirable safety with cellular viabilities exceeding 80% at 50 μg/mL

and exceeding 60% even at a high concentration of 200 μg/mL. Comparing to

MWNT-COOH, enhanced biocompatibility of f-MWNT was clearly observed in all

testing concentrations presumably because PEI 1.8k altered the exposed surface of

MWNT-COOH and shielded extreme negative charges on MWNT-COOH which both

do harmful to cells [5].

Notablely, in high concentration above 100 μg/mL, f-MWNT with iRGD

modified were less toxicity than those without iRGD, indicating iRGD functionalized

f-MWNT had a better biocompatibility. Introduction of CD onto f-MWNT did not

induce cytotoxicity since CD is a non-cytotoxic drug. These facts from MTT assay

reminded surface functionalization of MWNT deeply influence its toxicity. Our

engineering approaches made f-MWNT biocompatible vectors being available for

further research.

Figure S7. Cell viabilities of (A) A549 and (B) HUEVC cells incubated with different

f-MWNT/pDNA complexes at various concentrations for 48 h, respectively. Data were shown as

mean ± S.D. (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001.

Western blot

Angiogenesis which is regulated by elaborate mechanism could be activated

through multiple pathways. Therein, VEGF is the most powerful regulator of

physiological and pathological angiogenesis contained in almost all approaches. By

detection of AT1R, AT2R and VEGF expression in protein level, it provided direct

proofs for the effect of complexes on AT1R and AT2R regulation, as well as the

relationship between angiotensin receptors and VEGF. Fig. S8 showed, when A549

presented in high AngII concentration (100 nM) as in vivo tumor environment [6, 7],

it remarkablely upregulated AT1R and VEGF expression with 1.8 and 3 folds

comparing to control group, respectively, but it showed no appreciable change on

AT2R expression. Sequentially treating with formulations contained CD, despite in

conjunct form or a simple mixture, resluted an antagonism to AngII with concomitant

decrease of AT1R and VEGF. It was not hard to surmise that CD as an ARB which

prevented AT1R from AngII activation reversed the upregulation. On the other hand,

transfect cells with AT2 gene also received a decline of VEGF expression. With

overexpression of AT2R, AngII induced AT2R activation could be enhanced which in

turn leaded to a reported negative regulation of VEGF. Although it seems no direct

relationship between AT1R and AT2R expression, we were delighted to find that

combination of CD and AT2 gene synergistically led to VEGF downregulation.

Actually, VEGF proteins were even lower than the basal level in no AngII treated

cells. The above results highlight the prospect of simultaneous conducting AT1R and

AT2R in antiangiogenic therapy, especially in some AngII excess lesions. Exploiting

the high AngII condition in lung cancer, the strategy to concurrently block AT1R and

increase AT2R probably inhibit tumor progression by significantly lowering VEGF

level.

Figure S8. The effects of different formulations on VEGF, AT2R and AT1R protein expression in

A549. (A) Representative immunoblots of VEGF, AT2R and AT1R proteins in A549 cells after

treated with different formulations for 48h. All cells were pretreated with Ang II (100 nM) which

was utilized to simulate in vivo tumor microenvironment with high Ang II concentration except

control groups without any treatment. The corresponding quantitative results of (B) VEGF, (C)

AT2R and (D) AT1R protein expression in A549 after treated with different formulations were

normalized to control groups and expressed as mean ± S.D. (n = 3). *P < 0.05, **P < 0.01 and

***P < 0.001.

TUNEL assay

The results of TUNEL assay showed our complexes effectively induce apoptosis

in tumor (Fig. S9). 1.4 folds of TUNEL-positive cells was detected in iRGD-PEI-

MWNT-SS-CD/pAT2 group comparing to control, indicating that blocking of AT1R

slightly induce apoptosis of tumor cell. However, delivering pAT2 to tumor received

around 3 times of TUNEL-positive cells comparing to control. The data revealed that

up-regulation of AT2R was a powerful strategy to induce apoptosis of tumor. Our

complexes delivering CD and pAT2 effectively also suppressed tumor growth by

inducing apoptotic cell death of tumor.

Figure 9. In vivo TUNEL assay. (A) Representative fluorescence microscopic images (100×) of ex

vivo tumor sections from A549 bearing nude mice after 14 days injection of different

formulations. The nuclei were stained with DAPI (blue) and apoptotic cells were labeled with

fluorescein-dUTP (green). (B) Quantification of apoptotic cells in A549 bearing nude mice after

14 days injection of different formulations. Results were quantified as fold change to control. All

results above were expressed as mean ± S.D. (n = 7). **P < 0.01 and ***P < 0.001.

In vivo safety evaluation

Figure 10. In vivo safety evaluation. (A) In vivo toxicity examined of

iRGD-PEI-MWNT-SS-CD/pAT2 complexes by hematology analysis. Results were expressed as

mean ± S.D. (n = 7). (B) Representative images (200 ×) of histological analysis of major organs in

mice treated with saline and iRGD-PEI-MWNT-SS-CD/pAT2.

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