Facile Engineering of Indomethacin-Induced Paclitaxel Nanocrystal

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Biological and Medical Applications of Materials and Interfaces

Facile Engineering Indomethacin Induced Paclitaxel Nanocrystal Aggregates as Carrier-Free Nanomedicine with Improved Synergetic Antitumor Activity Chengyuan Zhang, Ling Long, Yao Xiong, Chenping Wang, Cuiping Peng, Yuchuan Yuan, Zhirui Liu, Yongyao Lin, Yi Jia, Xing Zhou, and Xiaohui Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22336 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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ACS Applied Materials & Interfaces

Facile Engineering Indomethacin Induced Paclitaxel Nanocrystal Aggregates as Carrier-Free Nanomedicine with Improved Synergetic Antitumor Activity Chengyuan Zhang 1, 2, ‡, Ling Long1, 3, ‡, Yao Xiong1, 4, Chenping Wang1, Cuiping Peng1, Yuchuan Yuan1, Zhirui Liu1, 5, Yongyao Lin1, Yi Jia1, *, Xing Zhou2, *, Xiaohui Li1, *

1 Department of Pharmaceutics, College of Pharmacy, Third

Military Medical University, Chongqing 400038, China 2 School of Pharmacy and Bioengineering, Chongqing University of

Technology, Chongqing 400054, China 3 Department of Oncology, Xinqiao Hospital, Third Military

Medical University, Chongqing 400042, China

ACS Paragon Plus Environment

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4 Institute of Pharmacy, Pharmaceutical College of Henan

University, Kaifeng 475004, China 5 Department of Pharmaceutics, Southwest Hospital, Third Military

Medical University, Chongqing 400038, China ‡ These authors contributed equally to this work. * To whom correspondence should be addressed. Yi Jia ([email protected]), Xing Zhou ([email protected]), and Xiaohui Li ([email protected]) KEYWORDS: Nanocrystal aggregates, Nanomedicine, Self-assembly, Carrier-free, Synergetic antitumor effects

ABSTRACT:

Carrier-free

nanomedicines

mainly

composed

by

nanocrystals of drug are considered as promising candidates for the

next

generation

of

nanodrug

formulations.

However,

such

nanomedicines still need to be stabilized by additive surfactants, synthetic

polymers

or

Contributed by the strong

biologically-based

macromolecules.

intermolecular interactions between

indomethacin (IDM, a COX-2 inhibitor) and paclitaxel (PTX, a chemotherapy drug), we herein successfully engineered a novel kind of carrier-free nanomedicines that organized as IDM induced PTX nanocrystal aggregates via one-pot self-assembly without any nonactive excipients. In the assemblies of IDM and PTX (IDM/PTX


2

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assemblies),

PTX

molecules, like

nanocrystals

are

casted

with

amorphous

IDM

“brick-cements” architecture. In serum, these

nanoassemblies could rapidly collapsed into a great number of smaller nanoparticles, thus targeting the tumor site through EPR effect. Under the assistance of IDM on immunotherapy, the IDM/PTX assemblies showed obviously improved synergetic antitumor effects of

immunotherapy

and

chemotherapy.

The

self-assembly

of

two

synergistic active substances into nanomedicines without any nonactive

excipients

might

open

an

alternative

avenue

and

give

inspiration to fabricate novel carrier-free nanomedicines in many fields.

INTRODUCTION Chemotherapy is still the most important strategy in tumor therapy by killing cells derectily or inhibiting proliferation of tumor cells.

Most

of

chemotherapy

agents

have

very

low

aqueous

solubility, which hampered their clinical application greatly. With

the

varieties

rapid of

polymer-drug

development

nanoscale

of

delivery

conjugates4-5,

nanotechnology1, tools

polymeric

including

micelles6-7

tremendous

liposomes2-3, and

protein-

based nanoparticles8 have been developed for carrying hydrophobic


3


drugs

to

lesions9-10.

Although

these

Page 4 of 54

tools

have

significantly

improved aqueous dispersity, biodistribution, bioavailability and targeting ability of drugs11, most of nanomedicines are still not suitable for clinical applications due to their relatively low drug loading, and potential undesired side effects derived from non-active excipients12-13, which were utilized to construct these delivery systems. On the other hand, carrier-free nanomedicines engineered with hydrophobic drug entirely can provide ultra-high drug loading and avoid undesired side effects discussed above, which has been developed as an emerging promising platform for drug delivery14-16. However, such nanomedicines still need to be stabilized

by

additive

surfactants,

synthetic

polymers

or

biologically-based macromolecules to avert further growth into larger

crystals

or

agglomerates.

Meanwhile,

the

shape

and

structure of this kind of nanomedicines are not tunable commonly1718.

Therefore,

to

form

nanomedicines

without

any

non-active

excipients still needs further breakthroughs. Another obstacles for clinical application of chemotherapy is that the fast growth of tumor is not only promoted by disordered proliferation

of

microenvironnement

tumor and

cells, tumor

but immune

also

supported

escape.

by

Therefore,

tumor the

combination of immunotherapy and chemotherapy has gradually become


4

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a new direction in cancer treatment. Prostaglandin E2 (PGE2), which is synthesized in lymphoid organs as well as the tumor site, play a key role in generation of immune suppressive microenvironment of tumor19. Contributed to the regulation of PGE2, monocytes preferred to be alternatively activated into anti-inflammation macrophages, also called as M2 macrophages, for promoting tumor immune escape via

secreting

anti-inflammatory

cytokines20-21.

Indomethacin

(IDM), a nonsteroidal anti-inflammatory drug that inhibit the synthesis of PGE2, has ability of reducing the PGE2-mediated M2 polarization

of

macrophages

and

increasing

pro-inflammation

macrophages (M1 phenotype) ratio in tumor microenvironment and enhancing immune response on tumor cells20, 22. Furthermore, IDM can also sensitize resistant transformed tumor cells to macrophage cytotoxicity22. In addition to local immunoregulation, IDM has been proved

to

smartly

instruct

myeloid-derived

suppressor

cells

(MDSCs) in whole body to enhance suppressive activity in autoimmune inflammation,

but

reduced

suppressive

activity

as

a

dominant

proinflammatory profile to foster an efficient anti-tumor immune response in tumor microenvironment23-25. Based on the discussion above, IDM has great potential to be employed as an immunotherapy agent to combine with chemotherapy for treating tumors.


5


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In our previous studies26-28, we found that there was strong intermolecular interaction between IDM and Paclitaxel (PTX, a widely applied chemotherapy agent). Therefore, we hypothesized that the intermolecular interaction between IDM and PTX would support to construct a brand-new kind of carrier-free nanomedicine without

any

other

inactive

substances.

Based

on

that,

we

successfully prepared carrier-free nanomedicine composed by PTX and IDM, denoted as IDM/PTX assemblies, via one-pot self-assembly with tunable morphology (Figure 1a). The architecture of IDM/PTX assemblies was revealed that paclitaxel nanocrystals were casted with amorphous IDM as “brick-cements”. In serum, the IDM/PTX assemblies could rapidly release PTX nanocrystals and free IDM molecules. The released paclitaxel nanocrystals could specifically reach lesions of tumor by enhanced permeability and retention (EPR) effect for directly inhibiting the growth of tumor, meanwhile, the released IDM molecules could regulate the balance of immunity in the whole body for inhibiting tumor immune escape. Thus, the IDM/PTX assemblies showed obviously improved synergetic antitumor activities

of

immunoregulation

and

chemotherapy,

compared

to

simply combined administration of IDM and PTX25. As one brand-new kind of carrier-free nanomedicine, the IDM/PTX assemblies were entirely composed of active drugs with no non-active excipients or materials participated, which could provide perfect drug loading


6

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and avoid undesired side effects from non-active excipients or materials.

Figure 1 Schematic illustration and morphology of the IDM/PTX assemblies. (a) General procedure for the fabrication of the IDM/PTX assemblies as paclitaxel nanocrystal aggregates; (b) The 2D conformation of IDM/PTX complex calculated by Autodock 4.2; TEM (c) and SEM (d) images showing sphere-like IDM/PTX assemblies at feed weight ratio of 2/1, scale bars represent 500 nm; (e) CLSM image of IDM/PTX assemblies at feed weight ratio of 2/1 indicating a uniform thin shell of IDM. Scale bars represent 500 nm. MATERIALS AND METHOD Materials: CdSe/ZnS quantum dots (QDs) with an emission wavelength at

620

nm

was

purchased

from

Ocean


NanoTech,

LLC

(USA).

7


Indomethacin

(IDM),

paclitaxel

(PTX),

Page 8 of 54

docetaxel

(DTX),

were

obtained from Sigma-Aldrich (USA). 4’, 6-Diamidino-2-phenylindole (DAPI)

were

purchased

from

Invitrogen

(USA).

Penicillin,

streptomycin, and fetal bovine serum (FBS) were purchased from Gibco (USA). MDA-MB-231 and MCF-7 human breast cancer cell lines were purchased from Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China). DMEM medium was obtained from HyClone (USA). Female BALB/c athymic nude mice (4-5 w, 16-18 g) were obtained from the Animal Center of Third Military Medical University. CD86, CD68, CD206, iNOS, TUNEL and Ki-67 antibodies were

purchased from

Abcam

(USA).

All

the other reagents are

commercially available and used as received. Preparation

of

assemblies,

a

IDM/PTX dialysis

assemblies. procedure

To was

construct performed.

IDM/PTX Briefly,

indomethacin (IDM) and paclitaxel (PTX) at different weight ratios were dissolved in DMSO. The obtained solution was then dialyzed against deionized water at 25 °C. The outer aqueous solution was exchanged every 2 h. After 24 h of dialysis, samples were collected for analysis without further treatment. The drugs contents were determined by high-performance liquid chromatography (HPLC; LC20A, Shimadzu, C8 column 4.6×150 mm. Mobile phase A: water with 0.02 % acetic acid, 45 %; Mobile phase B: acetonitrile, 55 %. Flow rate was 1 mL/min and analysis time was 15 mins), and the drug


8

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loading

content

and

entrapment

efficiency

were

calculated

according to the following equations:

Loading content (%) =

Entrapment efficiency =

The weight of drug in nanoassemblies (mg) 100% (1) The weight of nanoassemblies (mg)

Drug content in nanoassemblies (%) 100% Theoretical drug content (%)

(2)

Characterization of various particles. The particle ζ-potential, hydrodynamic diameter and distribution were measured by a Malvern Zetasizer Nano ZS instrument at 25°C. Scanning electron microscopy (SEM) was conducted on a FIB-SEM microscope (Crossbeam 340, Zeiss). Transmission electron microscopy (TEM) observation was carried out on a Tecnai-10 microscope (Philips, the Netherlands) operating at an acceleration voltage of 80 kV, for which samples were carefully placed onto the carbon-coated copper grids. A high-resolution field emission analytical TEM JEOL 2010F with 200 kV and 0.24 nm point resolution was used for the High-resolution TEM (HR-TEM) study. Confocal laser scanning microscopy (CLSM) observation was performed using a Zeiss LSM510 laser scanning confocal microscope. Super-resolution imaging was performed using a DeltaVison OMX Blaze

microscope

(GE

Healthcare,

USA).

Differential

scanning

calorimetric (DSC) measurements were performed by Perkin Elmer Instruments (Pyris Diamond series). The samples were heated from 20 to 250 oC at a constant rate of 10 oC min-1 under N2 atmosphere.


9


The

X-ray

PANalytical

diffraction X’Pert

(XRD)

Powder

patterns instrument

Page 10 of 54

were

collected

under

the

on

a

following

conditions: scanned from 3° to 60° (2θ value) at a scanning speed of 5° min−1. X-ray scattering measurements were performed using a SAXSess MC2 high flux small angle X-ray scattering instrument (Anton Paar, Austria, Cu-Ka, λ= 0.154 nm), equipped with a Kratky slits system in transmission mode. FT-IR spectra were acquired on a Perkin-Elmer FT-IR spectrometer (100S). Absorption spectra was recorded on Pgeneral TU-19 spectrometer using a pair of integral spheres. In vitro release experiments. The in vitro release experiments were carried out via dialysis method. 1 mL IDM/PTX assemblies suspension was put into a dialysis membrane (MWCO 1000 Da). Mixture suspension of PTX and IDM was taken as control. The dialysis membranes were immersed into vials containing 25 mL of PBS in a shaking bed at 37 ◦C. Tween 80 (0.1% v/v) was added into the release medium to improve the solubility of PTX and IDM in PBS solution (pH 7.4, 0.01 M). The amount of released each drug was detected by HPLC. Computational simulations. The multiscale simulation methodology adopted here synergizes both molecular dock and DPD techniques. Interaction modeling

energy

using

were

AutoDock

theoretically software

assessed

package


by

(AutoDock

molecular 4.2,

the

10

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Scripps Research Institute). To further validate this point, DPD was used to simulate the formation of IDM/PTX assemblies. PTX, IDM and water molecules were firstly coarse-grained to six, three and one beads, respectively (Figure S1). With a fully atomistic detail, Blends module incorporated in the Materials Studio 2017 software (Accelrys Inc, kindly provided by Analytical& Testing Center of Sichuan university) was employed to calculate the Flory-Huggins interaction parameters between binary components (Table S1); On this basis, DPD simulations were applied, at a coarse-grained level, to investigate the assembly process of IDM/PTX assemblies with various morphologies. Because the morphologies and aggregate sizes are almost the same in the box of 100×100×100 rc3 and 200×200×200 rc3 (Figure S2), a box of 100×100×100 rc3 was employed to simulate the morphology of IDM/PTX assemblies, and a box of 200×200×200 rc3 was employed to simulate the dynamic formation process of IDM/PTX assemblies. The detailed simulation parameters are given in Supplementary methods. Intracellular uptake. For the cellular uptake study, MDA-MB-231 cells (Human mammary cancer cells) were seeded in a 24-well plate at a density of 1.0 × 105 cells per well in 500 μL growth medium. IDM/PTX assemblies or Raw PTX at 8.5 μg/mL of PTX were added to the dishes. The cells were then incubated for 2, 4 and 8 h. Before observation, the cells were washed three times with PBS and then


11


Page 12 of 54

lysed. The drug content in cells was measured by HPLC, while the content of total proteins was quantified by a BCA method. The content of cellular drug was normalized to the protein content. Cell viability assays. MDA-MB-231 cells and MCF-7 cells were seeded in a 96-well plate at a density of 1 ×104 cells well-1 and cultured in DMEM supplemented with 10% FBS and 2 mM GlutaMAX at 37 °C in 5% CO2, respectively. The cells were exposed to varying amounts of IDM/PTX assemblies or PTX at different doses of PTX in a total volume of 200 μL for 48 h. After aspirating the supernatant of each well and washing three times with PBS. The cell viability was quantified by CCK-8 method. After removal of the medium, 100 μL 10% CCK-8/ RPMI 1640 (v/v) per well was added for another 2 h incubation. The 450 nm absorbance of each wells were then measured (ThermoFisher

SCIENTIFIC).

quadruplicate

and

the

All

relative

experiments cell

were

viability

performed

was

in

normalized

relative to the untreated control cells. Animal experiments. All the animal care and experimental protocols were performed in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of China (No. 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of Third Military Medical University. Female BALB/c athymic nude mice (4-5 w, 16-18 g) were obtained from the Animal Center of Third Military Medical University. Animals were housed in standard cages


12

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under conditions of optimum light (12:12 h light-dark cycle), temperature (22 ± 1 °C) and humidity (50-60%), with ad libitum access to water and food. All the animals were acclimatized to the laboratory for at least 7 days before experiments. In vivo antitumor study. Female BALB/c athymic nude mice (weighing 18-22 g, 4 weeks old) were acclimatized for 7 days after arrival. The MDA-MB-231 xenografts model were established by subcutaneous inoculation on the back with a fragment of MDA-MB-231 tumor via 20-gauge needle from an in vivo passage. When the tumors reached an average volume of 100 mm3, mice were randomized into four groups of 5 mice each. Saline, PTX, mixture of IDM and PTX, and IDM/PTX assemblies

were

injected

intravenously

for

every

four

days,

respectively. For all PTX-containing formulations, the PTX dosage was kept at 5 mg kg-1. The tumor size was measured at administrate time points. Tumor volume was estimated as ellipsoid sphere (V=a × b2/2), where a and b refer to the major and minor axes of the tumor

measured

by

a

caliper,

respectively.

Those

mice

were

sacrificed after seventeen days treatment. The tumors of mice were weighed and the tumor growth inhibition (TGI= (1-(Mean tumor weight of treatment group)/ (Mean tumor weight of control group)) ×100%) was calculated. All tumor tissues were excised and divided into two parts. One part was collected for immunofluorescence assay, the other one was made into tissue homogenate for evaluating of


13


Page 14 of 54

the concentration of PTX in tumor and expression of tumor necrosis factor-α (TNF-α) and IL-10. The main organs were excised and fixed in 4% paraformaldehyde, and then pathological sections were made and stained with hematoxylin and eosin (H & E). RESULTS AND DISCUSSION Preparation and characterization of IDM/PTX assemblies. Calculated by Autodock 4.2 and Materials studio 2017, we found that there was a strong interaction force and great miscibility between

IDM

and

PTX29,

which

was

contributed

by

multiple

synergistic forces between them, including π-π stacking, H-bonding interaction and hydrophobic interaction (Figure 1b, and S3a, b). And

forced

by

these

multiple

synergistic

intermolecular

interactions, free IDM in aqueous solution was adsorbed to the surface of PTX crystals, supported by the phenomenon that the concentration of IDM in aqueous solution was decreased in the presence of PTX crystal (Figure S3c), which suggested that IDM had the ability to assemble with PTX crystal. Within

this

mind,

IDM/PTX

assemblies

were

prepared

readily

through a one-pot assembly with IDM/PTX feed weight ratio of 2/1 at PTX initial concentration of 5 mg/mL. Well-defined spherical structures with mean size of 461.6 ± 57.72 nm were observed (Figure S4a) with no sight of typical PTX or IDM crystals, which confirmed by transmission electron microscopy (TEM) and scanning electron


14

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microscopy (SEM) images (Figure 1c, d, and S4c-e). Based on the fluorescence of IDM, the fluorescent image under confocal laser scanning microscopy (CLSM) revealed a uniform thin shell of IDM around the central core, which was conjectured to be the PTX nanocore (Figure 1e). Zeta potential measurements revealed that these assemblies had negative surface charge (-25.5 ± 0.702 mV) close to raw IDM (-22.2 ± 3.96 mV) (Figure S4b), suggesting that the surface of IDM/PTX assemblies was mostly composed by IDM.


15


Page 16 of 54

Figure 2 Tunable morphology of IDM/PTX assemblies. (a) Schematic, TEM, SEM and CLSM images of IDM/PTX assemblies at PTX concentration of 5 mg/mL, with various feed weight ratio of IDM/PTX, scale bars represent 500 nm; (b) Schematic, TEM, SEM and CLSM images of IDM/PTX assemblies at various PTX concentration, with IDM/PTX feed weight ratio of 2/1, scale bars represent 500 nm. DSC curves (c) and X-ray diffraction patterns (d) of IDM/PTX assemblies at PTX concentration

of 5

mg/mL,

with

various

feed weight ratio of

IDM/PTX, as well as IDM and PTX crystals dialyzed from water ; (e) HR-TEM and SAED pattern of the IDM/PTX assemblies at feed weight ratio of 2/1 and initial PTX concentration of 20 mg/mL; (f) Orientation-averaged plot of the scattering intensity versus the modulus of the scattering vector Q recorded for powdered samples of typical PTX crystal (blue circles) and IDM/PTX assemblies (pink circles) performed on SAXS. The proﬁles of IDM/PTX assemblies provided indications for structural complexity at the length scale of nanometers. Interestingly, by varying the weight ratio of IDM/PTX molecules, hydrophilic/hydrophobic

balance

of

the

IDM/PTX

system

may

be

regulated, thereby modulating the shape and size distribution of resulting assemblies (Figure 2a, b and S5). For instance, wirelike,

net-like,

honeycomb-like,

sphere-like,

and

capsule-like

assemblies without typical crystalline PTX or IDM were obtained


16

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respectively (Figure 2a), along with the ratio of IDM/PTX altered from 1/4, 1/2, 1/1,1/0.5 to 1/0.25 at a PTX initial concentration of 5 mg/mL. On the other hand, for a given ratio of IDM/PTX at 2/1, we found that the shape of IDM/PTX assemblies was gradually changed from sphere-like to capsule-like and lamella-like (Figure 2b) along with the concentration of PTX rising from 5 to 20 mg/mL. These results suggested the predominant role of the initial drug concentration and weight ratio of IDM/PTX for controlling the final shape of IDM/PTX assemblies. As

we

know,

amount

of

studies

have

successfully

engineered

cocrystals or amorphous assemblies of different organic molecules through supramolecular chemistry30. To determine whether these IDM/PTX assemblies were formed as drug eutectic or amorphous formation, differential scanning calorimetric (DSC) and X-ray diffraction (XRD) measurements were carried out. It was amazing that only the crystalline peak (at nearly 235 oC) of PTX was represent in DSC results at all initial IDM/PTX weight ratios, without peak of IDM (Figure 2c). This indicated that there were PTX crystals in IDM/PTX assemblies, but no IDM crystals in IDM/PTX assemblies, so the IDM/PTX assemblies must not be a simple mixture similar to the physical way. Meanwhile, the IDM/PTX assemblies at each initial IDM/PTX weight ratio shared a similar XRD spectrogram to that of typical PTX crystal (Figure 2d). However, the percentage


17


Page 18 of 54

of amorphous in these assemblies was increased with the increase of the IDM mass ratio, as indicated by increased intensity of halo peaks in the XRD spectrograms (Figure 2d). Significantly, PTX in these assemblies was mainly existed as crystal, but the question that whether IDM was retained in these assemblies as amorphous form or not was still uncertain. For this reason, we measured the content of IDM in these assemblies further. With the increase of IDM mass ratio, the increased IDM loading clearly supported the inference that IDM must be retained as amorphous form in these assemblies (Figure S6). Under HR-TEM observation of the lamellalike

IDM/PTX

nanocrystals

assemblies, with

it

different

was grain

further

revealed

orientation

were

that casted

the in

amorphous substrate, and the selected area electron diffraction (SAED) pattern showed a polycrystalline diffraction ring (Figure 2e, and S7a), indicating that these amorphous substrates were probably composed by IDM, and the crystalline regions were PTX nanocrystals. The polycrystalline diffraction ring suggested that the arrangement of PTX nanocrystals in IDM/PTX assemblies was disordered. Furthermore, small-angle X-ray scattering (SAXS) was used to observe the internal structures of the IDM/PTX assemblies (Figure

2f).

Different

with

typical

PTX

crystal

with

little

structural complexity, the curve of IDM/PTX assemblies samples gave rise to several bent shapes, owing to smaller nanostructure features within the particles. Intensive positive deviation from


18

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Porod’s law indicated that there was density fluctuation within the IDM/PTX assemblies (Figure S7b). Further evidence proving that the IDM/PTX assemblies was formed as IDM induced PTX nanocrystal aggregates (iPNAs) with a “brick-cement” architecture was found from the unevenly distributed fluorescence of IDM in capsule-like IDM/PTX assemblies obtained by ultrahigh resolution microscope (Figure 3a). To further verify that IDM was amorphously distributed in IDM/PTX assemblies, the solid powder fluorescence spectrums of raw IDM, raw PTX, mixture of IDM and PTX, and IDM/PTX assemblies at various weight ratio of IDM/PTX were measured (Figure 3b and S8a). When IDM was transformed from crystal into amorphous form, the fluorescent maximum emission of IDM would be red shifted31. In consequence,

we

found

the

emission

wavelengths

of

IDM/PTX

assemblies red shifted from 460 nm to 500 nm, compared to IDM crystal

(Figure

3c

and

S8b),

which

further

demonstrated

the

amorphous form of IDM in the IDM/PTX assemblies. Moreover, the excitation wavelength of IDM/PTX assemblies was moved from 360 nm (assigned

to

IDM

crystal)

to

280

nm

(Figure

3c

and

S8c),

contributed by the FRET between IDM and PTX induced by their nanoscale spacing. Since the emission spectra of IDM and PTX are overlapped at DAPI area (Figure S8d), dramatically fluorescence enhanced

phenomenon

with

increasing

laser

exposure

time

was

interestingly observed, after IDM/PTX assemblies being exposed under CLSM for 5 minutes (Figure 3d and S8e). This interesting


19


result

might

be

induced

by

the

gradually

Page 20 of 54

quenching

of

PTX

fluorescence and the more directly exciting the fluorescence of IDM.

Figure 3 The brick-cements architecture of IDM/PTX assemblies. (a) The distribution and existent morphology of IDM in capsule-like IDM/PTX assemblies at PTX concentration of 10 mg/mL, with IDM/PTX feed weight ratio of 2/1, obtained by ultrahigh solution microscope. Scale bars represent 500 nm; (b) 3D fluorescence spectrum of IDM/PTX assemblies at PTX concentration of 5 mg/mL, with IDM/PTX feed weight ratio of 2/1. (c) Normalized fluorescence spectra of IDM/PTX assemblies and mixture of IDM and PTX. (d) The change of fluorescence intensity with laser channel of DPAI under CLSM in 20 mins. The IDM/PTX feed weight ratio of IDM/PTX assemblies employed in (c) and (d) is 2/1, at initial PTX concentration of 5 mg/mL. (e) The morphological changes of IDM/PTX assemblies with the


20

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removal of IDM by PBS (pH 9.0), monitored by TEM. The IDM/PTX feed weight ratio of sphere-like and capsule-like IDM/PTX assemblies are 2/1, at initial PTX concentration of 5 and 10 mg/mL. Scale bars = 500 nm. For further investigating the architecture of IDM/PTX assemblies, we added the assemblies into alkaline PBS (pH 9.0, 0.1 M-1) to remove IDM only. The kinetic morphologic deformations of spherelike and capsule-like IDM/PTX assemblies in process of IDM removal, were monitored under TEM (Figure 3e). For sphere-like IDM/PTX assemblies,

small

fragments

were

firstly

released,

and

then

reassembled to nets with the residual IDM. After IDM was entirely removed,

retained

PTX

fragments

were

regrew

to

typical

PTX

crystals. In case of capsule-like IDM/PTX assemblies, the smooth surface

of

microcapsules

was

firstly

changed

into

porous

structure, then crushed to porous lamellas, and finally regrew to typical

PTX

crystals.

These

results

demonstrated

that

PTX

nanocrystals were casted with IDM molecules again, and the very important role of IDM in maintaining the “brick-cement”-like iPNAs architecture of IDM/PTX assemblies. π-π stacking interaction forced the unique “brick-cement”-like architecture of IDM/PTX assemblies. UV-Vis and Fourier transform infrared (FT-IR) spectroscopy were used to investigate the driving force for formation of the unique


21


“brick-cement”-like

architecture

of

Page 22 of 54

IDM/PTX

assemblies.

In

comparison to individual IDM and PTX, as well as mixtures of IDM and PTX, the UV-Vis absorption spectra of IDM/PTX assemblies revealed a new absorbance band at 278 nm, which was 40 nm longer than the characteristic absorption of typical crystal of PTX (λ=237 nm), corresponding to the π-π stacking interaction between IDM and PTX (Figure 4a, and S9). It was noted that the intensity ratio of absorption band at 237 nm to 278 nm decreased with the increase of initial IDM/PTX weight ratio, indicating that the contact area of IDM/PTX increased with the initial IDM/PTX weight ratio increasing (Figure 4a). Meanwhile, FT-IR spectroscopy analyses of typical crystal of each drug and IDM/PTX assemblies were also performed. The peak at 1444 cm-1 of IDM correspond to the C-H out-of-plane motion of the indole ring, and the strong absorption bands of IDM and PTX at 1488 cm-1 are the results of the C-H out-of-plane motion of

the

phenyl

ring

(Figure

4b).

After

formation

of

IDM/PTX

assemblies, the strong absorption band 1488 cm-1 was red shifted to 1462 cm-1, which clearly indicated the π-π stacking interaction between IDM and PTX. Meanwhile, the disappearance of the absorption peak at 1444 cm-1 was owned to the electron sharing between indole ring and the benzene rings by π-π stacking (Figure 4b). There was no H-bonding formed in IDM/PTX assemblies that the stretching vibrations of carboxylic acids (922 cm-1 and 1690 cm-1) and C=O (1740


22

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cm-1) were not shifted compared to IDM and PTX crystals. To further confirm the importance of π-π stacking interaction in the formation of nanocrystal aggregates architecture, docetaxel (DTX), a similar structure of PTX, was employed. Because the number of phenyls in DTX is one less than PTX, the intermolecular energy between IDM/DTX was lower by 10% than that between IDM/PTX, which resulted a poor miscibility of DTX and IDM (ΔEmix =10.46782). As a result, we found respective DTX and IDM crystals were growing when they dialyzed together to against distilled water (Figure S10). These results illustrated that the main interaction contributed to drive the nanocrystal

aggregates

form

of

IDM/PTX

assemblies

was

π-π

stacking, similar as the intermolecular interaction we calculated by Autodock 4.2 (Figure 1b).

Figure 4 The characterization of π-π stacking interaction in the IDM/PTX assemblies (a-b). UV-vis spectrum (a) and FT-IR spectrum


23


Page 24 of 54

(b) of IDM/PTX assemblies at PTX concentration of 5 mg/mL, with various feed weight ratio of IDM/PTX, as well as IDM crystal and PTX crystal dialyzed from water. Computational simulation of the formation of iPNAs architecture. Multiscale computational simulations synergized both molecular dock and DPD techniques were carried out to uncover the mechanism forcing

the

intermolecular

iPNAs

architecture

energy

(IE)

of

of

IDM/PTX

assemblies.

the

IDM/PTX

assemblies

The were

calculated by Autodock 4.2. It was noted that the value of IE between IDM and PTX was just lower than the IE between IDM and IDM, but higher than the IE between PTX and PTX (Figure 5a), which suggested that IDM tended to spontaneously pack to PTX, but PTX molecules had much more opportunity to pack with themselves rather than with IDM.

Hence, PTX nanocrystals would be initially formed

when DMSO solution of IDM and PTX dialyzed against distilled water, and then IDM spontaneously adsorbed to PTX nanocrystals, rather than formed as IDM crystals. To further validate this point, dissipative particle dynamics (DPD), introduced by Hoogerbrugge and Koelman32 in 1992 and revised by Espaňol and Warren33, was used to simulate the formation of the iPNAs architecture in IDM/PTX assemblies. As a result, five representative micellar morphologies were obtained by changing the weight ratio of IDM/PTX. The micellar morphology was changed from cylinders to nets, honeycomb, sphere,


24

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and further to lamellas with the increase of initial IDM/PTX weight ratio (Figure 5b), that were excellently consistent with our experimental findings at corresponding weight ratio (Figure 2a). Those experimental result validated our proposed computational model, which allowed us to investigate the internal structure of IDM/PTX assemblies and self-assembly process of those systems further. From the comparison between full microstructures and PTX distribution of IDM/PTX assemblies obtained from DPD stimulation, we were able to clearly observe the internal structure of IDM/PTX assemblies that PTX aggregates were casted by IDM molecules like “brick-cements” in various initial IDM/PTX weight ratios (Figure 5b). For

further

investigating

the

formation

mechanism

of

iPNAs

architecture of IDM/PTX assemblies, the detailed assembly process of lamella-like micelles and sphere-like micelles were recorded and analyzed at more fine simulation parameters (Figure 5c). We overserved that PTX nanocrystals were firstly formed at the very beginning

with

the

phase

separation,

due

to

the

strongly

hydrophilic of PTX molecules. After that, IDM molecules clustered around

the

PTX

nanocrystal

and

coalesced

together

to

small

particles, and further grew to larger segmented rod-like micelles. The

rod-like

micelles

sequentially

grew

to

spheres

with

PTX

nanocrystal at initial IDM/PTX weight ratio of 2/1, or lamella


25


Page 26 of 54

covered by IDM with dispersed PTX nanocrystals at initial IDM/PTX weight ratio of 4/1. By combining these findings with the classic theory of crystal growth that final crystalline particles were further grown from firstly formed small crystalline nuclei34, the formation process of IDM/PTX assemblies in the dialysis experiment became clear. PTX nanocrystals were firstly formed as crystalline nuclei in the preliminary exchanging of organic solvent and water, and IDM was dissolved at the same time due to its higher solubility in

water.

Contributed

by

the

strong

interaction

between

PTX

nanocrystals and IDM, IDM tend to cluster around PTX nanocrystal cores,

and

then

these

nanocrystals

further

grew

into

small

particles with the increasing ratio of water in solution. The particles amorphously assemble with each other under the cementlike action of IDM that early adsorbed around PTX nanocrystals, and

finally

built

nanoscale

crystal

aggregates

with

various

morphology decided by the weight ratio of IDM/PTX.


26

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Figure

5

Computational

architecture

in

simulation

IDM/PTX

of

assemblies.

the

formation

(a)

The

of

iPNAs

intermolecular

energies in IDM/PTX system. (b) Full microstructures of IDM/PTX assemblies (up) and PTX distribution in IDM/PTX assemblies (down) from DPD simulations at different IDM/PTX feed weight ratio. (c) The dynamic process of the formation of IDM/PTX assemblies under DPD simulation. PTX and IDM molecules were colored as blue and red, respectively. Interestingly,

in

view

of

the

capsule-like

architecture

of

IDM/PTX assemblies formed at specific weight ratios of IDM/PTX, we rationally assumed that the capsule constructed by IDM and PTX may be capable of serving as nanovehicles for other nanoparticles,


27


Page 28 of 54

such as quantum dots. For evidencing this hypothesis, CdSe/ZnS quantum dots (QDs, ~10 nm) with an emission wavelength at 620 nm were selected as the model nanoparticle (Figure S11a), which have potential applications in surveillance, machine vision, industrial inspection, spectroscopy, and fluorescent biomedical imaging35. QDs loaded IDM/PTX assemblies were successfully fabricated by onepot

assembly

at

the

IDM/PTX

ratio

of

2/1

with

initial

PTX

concentration of 10 mg/mL (Figure S11b). From TEM images, we observed that QDs were scattered in the microcapsule (Figure S11c). Similar as IDM/PTX assemblies, after the entirely removal of IDM by PBS (pH 9.0, 0.1 M-1), retained PTX fragments were regrew to typical PTX crystals and QDs were released (Figure S11d-e). These results illustrated the ability of IDM/PTX assemblies to load other nanoparticles and its potential in theranostic application.


28

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Figure 6 In vitro evaluation of IDM/PTX assemblies. (a) Comparison of the in vitro release behaviors of IDM between IDM/PTX assemblies (IDM/PTX = 2/1) and IDM crystal, n=3; (b) IDM/PTX assemblies were distributed in cell culture medium for 2 hours, a number of nanocrystals released from IDM/PTX assemblies. Scale bar = 500 nm; The TEM images (c) and (d) size distribution of IDM/PTX assemblies (2/1) after incubation in serum for 2 h and 8 h. Scale bars = 100 nm; Cellular uptake (e) and in vitro antitumor activity (f) of


29


Page 30 of 54

IDM/PTX assemblies in MDA-MB-231 cells. *, p < 0.05; ***, p < 0.001; compared with PTX; n=4. (g) The antitumor activity of various PTX formulations in MDA-MB-231 cells co-cultured with activated macrophage. *, p < 0.05, compared with M0 macrophage; #, p < 0.05, compared with M1 macrophage; %, p < 0.05, compared with raw PTX; ^, p < 0.05, compared with raw IDM; n=4. (h) The CD206 and iNOS expression of macrophage after treatment. Potential application of IDM/PTX assemblies as a brand-new kind of carrier-free nanotherapeutic. Considered with appropriate size for in vivo treatment and the safe window of IDM, IDM/PTX assemblies at IDM/PTX feed weight ratio of 2/1 with initial PTX concentration of 5 mg/mL was employed to evaluate the potential application of IDM/PTX assemblies as a brand-new kind of nanotherapeutic. As a nanotherapeutic, IDM/PTX assemblies was endowed with appropriate nanoscale size, a total 100% drug loading, and a perfect encapsulation efficiency (EE) and high

drug

loading

of PTX (Figure S5),

which

were

critically

important for drug delivery and effective anticancer therapy. In addition, after incubated in 5 % glucose solution for 24 hours, the

size

of

IDM/PTX

assemblies

was

barely

changed,

which

demonstrated the IDM/PTX assemblies were stable enough in isotonic conditions (Figure S12). Besides, amorphous IDM component was rapidly released in PBS (pH 7.4) (Figure 6a), while PTX component


30

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was remained for a long time in medium with sustained release profile (Figure S12), which was gifted by the unique “brickcement”-like

iPNAs

architecture

of

IDM/PTX

assemblies.

When

IDM/PTX assemblies were incubated in cell culture medium or serum for 2 hours, a great number of smaller nanoparticles released from IDM/PTX assemblies was observed clearly (Figure 6b, c). Notably, these smaller nanoparticles existed stably in serum for 8 hours (Figure 6c, d). Based on these above, we supposed that IDM could be rapidly released for regulating immunity system after the administration

of

IDM/PTX

assemblies,

while

PTX

nanocrystals

retained in released smaller nanoparticles targeted effectively toward tumor tissue due to the EPR effect and were uptaken by tumor cells for killing them further. In our in vitro antitumor evaluation, we found that cellular uptake efficiency of PTX was significantly promoted in IDM/PTX assemblies treated cells than free PTX treated ones, which might be benefited from the nanocrystal form of PTX (Figure 6e). In consequence, the IDM/PTX assemblies showed superior inhibition on the proliferation of MDA-MB-231 cells and MCF-7 cells than the free PTX after 48 h of incubation (Figure 6f and S14a). Despite that IDM showed no cytotoxicity for tumor cells at corresponding dose (Figure S14b), IDM was reported to drive macrophages towards the M1 phenotype and promote the cytotoxicity of macrophage to


31


Page 32 of 54

tumor cells22, 25. Hence, the in vitro anti-tumor effect of IDM/PTX assemblies on MDA-MB-231 cells co-cultured with macrophages in transwell was further evaluated (Figure S15). These macrophages were pre-polarized from THP-1 human monocyte cells to M1 or M0 macrophage, which was verified by the expression of iNOS (marker for M1 macrophage) and CD206 (marker for M2 macrophage).

Figure 7 Therapeutic effect of various PTX formulations in mice bearing MDA-MB-231 human mammary xenografts and representative


32

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immunofluorescence images of tumor tissues harvested at the end of study from mice receiving various treatment. (a) Relative tumor volume and images of tumor after excision. Red arrows in (a) denote the time to give administrations of various PTX formulations. n=5. Scale bar in (a) was 1 cm; (b) Tumor growth inhibition of various PTX formulations; (c) Representative immunofluorescence images of Ki-67-positive cells (red) and cells undergoing apoptosis (TUNEL stain; green), scale bars represent 50 μm; (d) The concentration of

PTX in

tumor

tissue.

**,

p

Facile Engineering of Indomethacin-Induced Paclitaxel Nanocrystal

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