Matrix metalloproteinase 2-sensitive multifunctional polymeric micelles for tumor-specific co-delivery of siRNA and hydrophobic drugs

HAL Id: hal-02995779

https://hal.archives-ouvertes.fr/hal-02995779

Submitted on 3 Dec 2020

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Matrix metalloproteinase 2-sensitive multifunctional polymeric micelles for tumor-specific co-delivery of

siRNA and hydrophobic drugs

Tao Wang, Lin Zhu, Federico Perche, Anton Taigind, Vladimir Torchilin

To cite this version:

Tao Wang, Lin Zhu, Federico Perche, Anton Taigind, Vladimir Torchilin. Matrix metalloproteinase 2- sensitive multifunctional polymeric micelles for tumor-specific co-delivery of siRNA and hydrophobic drugs. Biomaterials, Elsevier, 2014, 35 (13), pp.4213 - 4222. �10.1016/j.biomaterials.2014.01.060�.

�hal-02995779�

Matrix metalloproteinase 2-sensitive multifunctional polymeric micelles for tumor-specific co-delivery of siRNA and hydrophobic drugs

Lin Zhu, Federico Perche, Anton Taigind, and Vladimir P Torchilin Center for Pharmaceutical Biotechnology and Nanomedicine,

Northeastern University, Boston, MA 02115;

[email protected]

ABSTRACT SUMMARY

To deliver siRNAs and hydrophobic drugs, a novel matrix metalloprotease 2 (MMP2)-sensitive self-assembling copolymer, polyethylene glycol-polyethylenimine-1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (PEG-PEI-PE),

has been developed.

The micelles formed by PEG-PEI-PE possess several key features for siRNA and drug delivery, including (i) stability in physiological fluids thanks to the outer PEG “shell”; (ii) complexation of siRNA by PEI; (iii) drug- solubilization in the lipid “core”; (iv) passive tumor targeting via the EPR effect; (v) tumor targeting triggered by the MMP2-sensitivity; and (vi) tumor-cell selective endocytosis after MMP2-activated exposure of the previously hidden PEI block. (Fig. 1) These cooperative functions will ensure the improved tumor targetability and enhanced tumor cell internalization of co-loaded siRNAs and hydrophobic drugs.

Key words: polymeric micelle, self-assembly, tumor targeting, matrix metalloproteinase 2, co-delivery, siRNA.

INTRODUCTION

The therapeutic effects of siRNAs are significantly compromised by their poor stability, short circulation time, non-specific tissue distribution, and insufficient cellular uptake. PEI, a widely used gene delivery carrier, is able to condense siRNAs and interact with cell membranes via electrostatic interaction. In addition, its buffering capacity facilitates the endosomal escape of siRNAs and the resultant RNA interference. However, the non-specific interaction between PEI and cell membranes may lead to impaired tumor targeting and decrease siRNA’s therapeutic effect.

Paclitaxel (PTX), on the other hand, is one of the most commonly used antineoplastic agents. However, its applications are complicated by its low solubility, off-target toxicity and acquired drug resistance. Although various drug conjugates and polymeric micelles have been developed to solubilize and deliver PTX, co-delivery of hydrophobic drugs and siRNAs remains a challenge. Indeed, the combined use of siRNAs and chemotherapeutics is hindered by the current drug delivery technologies. Usually, because

of their distinct physicochemical properties, siRNAs and drugs are loaded into separate carriers for simultaneous administration. However, since these molecules may not be delivered to the same cell, low synergistic effects are observed.

MMP2, is known to be involved in cancer cells’

invasion, progression, and metastasis. The up-regulated MMP2 is considered as a biomarker for diagnostics and prognostics in many cancers, and also provides a strategy for tumor-targeted drug delivery via an enzyme-triggered mechanism

³

.

Based on the evidence of up-regulated Bcl2 (an anti- apoptotic protein) in cancer cells, correlated with resistance to paclitaxel treatment

¹

and sensitization to paclitaxel by anti Bcl2 siRNA (siBcl2)

²

, we proposed co-delivery of PTX and siBcl2 by a novel MMP2-sensitive self-assembling copolymer (PEG-PEI-PE). This novel polymer is based on the lipid-polymer PEI-DOPE (PEI-PE), recently designed by our group, which possessed the advantages of both PEI and DOPE and showed the improved efficiency of siRNA delivery

⁴

. PEG-PEI-PE self-assembles into a “core-shell”

structure in an aqueous environment (Fig. 1), able to load hydrophobic drugs. The presence of the MMP2-cleavable linker between PEI-PE and PEG allows targeting of aggressive tumor regions where MMP2 is up-regulated

³

.

Using the proposed polymeric micelle, both siBcl2 and PTX can be simultaneously and specifically delivered into tumor cells. The tumor cells will be efficiently killed by the synergism of therapeutics, resulting in improved anticancer efficacy and minimized toxicity. Here, we describe the synthesis and characterization of PEG-PEI-PE, siRNA polyplex formation, MMP2-dependent cellular uptake and in vitro co-delivery of siRNA and paclitaxel.

EXPERIMENTAL METHODS

The synthesis scheme is shown in Fig. 2A. First, the MMP2-cleavable peptide (GPLGIAGQ)

³

and PEG2000- NHS (1.2:1, molar ratio) were mixed in carbonate buffer (pH 8.5) at 4°C overnight, followed by dialysis (MWCO 2,000 Da) against water. Then, the branched PEI (1.8kDa) was reacted with N-glutaryl-DOPE at a 1:1 molar ratio to form PEI-PE

⁴

. Finally, PEG2000-peptide reacted with PEI-PE (1:1) in the presence of an excessive amount of NHS/EDC at RT overnight. The product was purified by dialysis (MWCO 8,000Da) and characterized by

¹

H-NMR. The critical micelle concentration (CMC) was determined using pyrene. The particle size and size distribution were determined by dynamic light scattering. To determine the MMP2- sensitivity, 1 mg/mL of PEG-PEI-PE was incubated with 5ng/µ?mL human MMP2 in HEPES-buffered saline (HBS) containing 10mM CaCl

₂

at 37°C overnight, followed by

++ +++

+ +

+ ++

++ ++ + ++++++++++

+ +

+ ++

++ +++ +++++ ++

+++ + +

+ ++

+ + +++

++ +++ + +

+ ++

++ +++

++++ +

++ ++ +

Cleavage by extracellular

MMP2

+ +

+++ + +

++

+ +++++++ +++ + + + ++

++ + ++

++ +++ +++++

+ + +++

+ + +++

+ + +++

+ + +++

Self-assembly

Complex formation

PEG(2000) MMP2-cleavable linker PEI(1800)

DOPE

siRNA Paclitaxel

+ + +++

+ + +++

+ + +++

Fig.1. The MMP2-sensitive nanocarrier for siRNA and drug delivery

analysis using thin layer chromatography (TLC), size exclusion HPLC, and zeta potential.

The siRNA polyplexes were formed by incubation of siRNA with PEG-PEI-PE in HBS for 20 min at RT at various Nitrogen to Phosphate (N/P) ratios. The complexes were examined by transmission electronic microscopy (TEM), light scattering, and gel retardation assay.

Cellular uptake and gene silencing of siRNA or siRNA/PTX containing nanopreparations were evaluated by fluorescence-activated cell sorting (FACS) and confocal microscopy after incubation in media containing 10% fetal bovine serum. All tests were performed with human non- small cell lung cancer (A549) cells.

RESULTS AND DISCUSSION

In the

¹

H- NMR (Fig. 2B), the peaks of hydrophobic

DOPE were

observed in chloroform but not in water suggesting the formation of a

“core-shell”

structure, which was further confirmed by their low CMC value (Fig. 2C).

The formed PEG-PEI-PE micelles had a small size in a broad range of pH indicating their stability against pH change during the preparation and administration.

The cleavage of the linker released the PEG block as evidenced by a new spot in the TLC and a new peak in size exclusion HPLC. PEG deshielding was further confirmed by the increase of the zeta potential from 26.8±2.4mV to 50.2±1.1mV after exposure of PEI block, increased charge promoting the cellular uptake of PEG-PEI- PE micelles by tumor cells (Fig. 3).

The siRNA/ PEG-PEI-PE complexes organized as uniform spherical nanoparticles were determined by TEM (Fig. 4A) The size of the siRNA polyplexes didn’t change before and after MMP2 cleavage, suggesting that the structure of the siRNA polyplexes was stable (Fig.

4B). At a N/P of 40, siRNA was completely complexed

(Fig. 4C) and protected from RNase III degradation (Fig.

4D). The FACS data showed that PEG-PEI-PE was able to efficiently transfer siRNAs into A549 cells in the presence of serum while no fluorescence was detected with uncleavable complexes, suggesting that the up-regulated MMP2 in tumor cells cleaved the peptide linker resulting in PEG deshielding and the resultant cell internalization (Fig. 5).

In addition to the loading of siRNAs, hydrophobic molecules such as paclitaxel can be loaded into the lipid core of PEG-

PEI-PE micelles.

Successful in vitro co- delivery of siRNA and paclitaxel was observed in almost all cells (98.2% cells positive for both siRNA and paclitaxel) with PEG-PEI-PE in serum-containing media whereas no co-delivery was observed with PEI 25kDa (Fig. 6A).

Intracellular co-delivery of siGLO Red siRNA and Oregon Green paclitaxel was confirmed by confocal microscopy (Fig. 6B).

CONCLUSION

The cationic PEG-PEI-PE self-assembles into micelles with a small size and uniform distribution, could co-load siRNA and hydrophobic paclitaxel. MMP2-cleavage could remove the PEG shell and expose the positively charged PEI, allowing cancer cell internalization.

REFERENCES

1. Padar, S.; van Breemen, C.; Thomas, D. W.;

Uchizono, J. A.; Livesey, J. C.; Rahimian, R. British journal of pharmacology 2004, 142, (2), 305-16.

2. Chen, A. M.; Zhang, M.; Wei, D.; Stueber, D.;

Taratula, O.; Minko, T.; He, H. Small 2009, 5, (23), 2673-7.

3. Zhu, L.; Kate, P.; Torchilin, V. P. ACS nano 2012, 6, (4), 3491-8.

4. Navarro, G.; Sawant, R. R.; Biswas, S.; Essex, S.;

Tros de Ilarduya, C.; Torchilin, V. P. Nanomedicine (Lond) 2012, 7, (1), 65-78.

ACKNOWLEDGMENT

This work was supported by the NIH Grant 1R01CA121838 in black to Vladimir P Torchilin.

Minutes

01234567891011121314151617181920

mAU

-2

02468

10 12 14 16 18 20

mAU

-2

02468

10 12 14 16 18 20 DAD-CH2 227 nm

PPPED

PEG-PEI-PE + MMP2

Minutes

01234567891011121314151617181920

mAU

-2

02468

10 12 14 16 18 20

mAU

-2

02468

10 12 14 16 18 20 DAD-CH2 227 nm

PPP r

PEG-PEI-PE

Fig. 3. The MMP2-sensitivity of PEG-PEI-PE. TLC (A), size exclusion-HPLC (B) and zeta potential (C).

PEG-GPLG

IAGQ-PEI

PEG-PEI-PE 50.7 4.2mV PEI-PE+PEG-peptide

PEG-PEI-PE+MMP2 PEI-PE

53.6 1.3 mV

26.8 2.4 mV 50.2 1.1 mV

A B C

EDC, NHS PEG2000-NHS

+

^H2N-GPLGIAGQ-COOH pH8.5, 4 С O

O NH2

C O N

O

O C O PEG H2 C

PEG2000-peptide MMP2 cleavable peptide

+

^PEI-PE

PEG-PEI-PE PEG-CONH-GPLGIAGQ-COOH

PEG-CONH-GPLGIAGQ-CONH-PEI

+

N–glutaryl-DOPE EDC, NHS

H P O^-O O O N O

O O

O H

O OH O

PEI (1800Da) Figure 2. Synthesis of MMP2-cleavable PEG-PEI-PE

PEG-PEI-PE_CDCL3_030112

12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

NormalizedIntensity

PPPU_D2O_030112

12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2

Chemical Shift (ppm) 0.1

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Normalized Intensity

CDCl3 D2O

PEIDOPE PEG TMS

Fig. 2. The synthesis scheme (A), ¹H-NMR (B), CMC (C) and particle size (D)of PEG-PEI-PE

pH Particle

size (nm)

PEG–PEI–

PE

5.5 16.7 3.7

7.4 16.5 5.1

9.0 21.7 5.4

PEI–PE 7.4 11.7 1.7

Table 1. The size of polymeric micelles

Figure 4: Critical micelle concentration (CMC) 0.82

0.84 0.86 0.88 0.9 0.92 0.94 0.96

-11 -10 -9 -8 -7 -6 -5 -4

I337/I334

Log C (M) PEI-DOPE

CMC: 8.32×10^-7M

0.82 0.84 0.86 0.88 0.9 0.92 0.94

-11-10 -9 -8 -7 -6 -5 -4

I337/I334

Log C (M) PEG-PEI-DOPE

CMC: 2.04×10^-7M

C D

PEG-PEI-PE

A B

Fig. 6. Co-delivery of siRNA (red) and paclitaxel (green).

FACS analysis (A) and Confocal microscopy (B).

B

Untreated cells

0.3% 0.5%

0%

PEI 25kDa

0%

0.3%

78.4%

PEG-PEI-PE 98.2%

0.4%

0.5%

A

DAPI

DIC Green (PTX) Red (siRNA) Merge

99.2% 21.3% 0.9%

siRNA

PEG-PEI-PE,

uncleavable PEG-PEI-PE

PEI-PE PEI 25K

PEG-PEI-PE PEG-PEI-PE,

uncleavable

Free siRNA PEI 25K PEI-PE

A

B

Fig. 5. Cellular uptake of siRNA (red) polyplexes in complete growth media (FACS (A) and confocal

microscopy (B))

10 20 40 PEG-PEI-PE

N/P siRNA

PEI-PE PEI

10 20 40 10 20 40

siRNA: 0.4ug Complex formation:

15min, RT

100nm

B

C A

D

N/P siRNA

siRNA/

PEG-PEI-PE 10 20 40

100 nm

A

0 50 100 150 200 250 300 350 400

size (nm) B

RNase III Heparin

PEG-PEI-PE

+ - + -

siRNA

+ - + -

C siRNA

siRNA/

PEG-PEI-PE RNase III

Heparin

- +

+ + - - + -

Fig. 4. The formation of siRNA complexes. The morphology (A), stability (B), siRNA condensation (C), and RNase III protection (D).

0 100 200 300 400

Size (nm) Polyplexes in HBS

PEI /siRNA

PEI-PE /siRNA

PEG-PEI-PE /siRNA

MMP2