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Self-Propelled and Targeted Drug Delivery of Polyaspartic Acid/Iron-Zinc Microrocket in the Stomach Minfeng Zhou, Ting Hou, Jinxing Li, Shanshan Yu, Zijian Xu, Miao Yin, Joseph Wang, and Xiaolei Wang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06773 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Self-Propelled and Targeted Drug Delivery of Polyaspartic Acid/Iron-Zinc Microrocket in the Stomach Minfeng Zhou, a† Ting Hou, a† Jinxing Li, b Shanshan Yu,a Zijian Xu,c Miao Yin,c Joseph Wang, b and Xiaolei Wang a* a College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China b Department of Nanoengineering, University of California San Diego, La Jolla, California 92093, United States c College of Life Sciences, Shandong Provincial Key Laboratory of Animal Resistance Biology, Shandong Normal University, Jinan 250014, China

† These authors contributed equally to this work. *Correspondence to: [email protected]

ABSTRACT: For many medical treatments, particularly cancer, it is necessary to develop a biocompatible microscale device that can carry a sufficient amount of a drug, deliver it to target sites. While chemically-powered micromotors have been applied in live animal therapy, many of them are difficult to biodegrade in vivo, which might cause toxicity and side effects. Here, we report on a microdevice that consists of a polyaspartic acid (PASP) microtube, a thin Fe intermediate layer, and a core of 1

ACS Paragon Plus Environment

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Zn. This device can be propelled using gastric acid as a fuel. After adsorbing doxorubicin onto a PASP surface, the microrocket can carry drugs, magnetically locate targets, permeate the gastric mucus gel layer, and increase drug retention in the stomach without inducing an obvious toxic reaction. All materials in microrockets are biocompatible and biodegradable and can be readily decomposed by the gastric acid or by proteases in the digestive tract. Such microrockets, made with polyamino acids, will extend the practical biomedical applications of micro/nanomotors. KEYWORDS: micromotors, microrockets, polyamino acid, stomach, drug delivery, self-propulsion

Gastric cancer is one of the most common cancers and frequently causes cancer-related deaths. Advanced gastric cancer with serosal invasion is usually unresectable, so the delivery of an anticancer drug to target sites in the stomach will be an effective and safe treatment.1 The ideal drug delivery system should carry a sufficient amount of a drug and accurately position them to the target sites. Recently, the nano/micromachines take the advantages of conventional nanocarriers (such as drug selectivity, biocompatibility and biodegradability), and their special function to swim efficiently and penetrate rapidly desired segments carefully controlled by external sources (such as light, ultrasound and magnetic field). Therefore, there is highly demand to develop a biocompatible microscale device that can be located in desired segments of the stomach and be actively trapped into the gastric mucus gel layer for prolonged retention.2 Recent efforts have also focused on the development of 2


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magnetic-propelled

biodegradable

micromachines

that

would

expand

their

applications in surgical treatments, diagnostics and drug-delivery techniques.3, 4 These micromachines always need special structures, such as artificial helical or flexible flagella, to make them move through the power of a rotating or oscillating magnetic field.5 On the other hand, chemically powered micromotors, which are based on converting chemical energy into propelling motion, represent an exciting biomedical tool for detoxification, motion-based sensing, and cargo transportation in environmental systems and living biosystems.6-8 Recent efforts have been devoted to exploring micromotors in live animals. For example, magnesium-based tubular micromotors (microrockets) coated with enteric polymer were precisely positioned and controllably retained in desired parts of the gastrointestinal (GI) tract to release the payloads.9 Magnetic propulsion and movement were applied to control the swarming of simulated bacterial flagella in vivo.10 The active drug-loaded magnesium micromotors were also applied to treat gastric bacterial infection in a mouse model.11 Because the drug was loaded in the poly (lactic-co-glycolic acid) layer, it should dissolve and be stable in ethyl acetate, which limited the usage of the method. Microrockets with acid/water-powered propulsion have shown effective movement in multiple bioenvironments, such as gastric acid and intestinal fluid.12, 13 In particular, to achieve their drug-delivery applications in vivo, the microrockets should possess good biocompatibility, low toxicity, and a zero-waste profile.14, However,

only

certain

kinds

(3,4-ethylenedioxy-thiophene),9

of

surface

reduced

15

materials

(polyaniline,16

poly

graphene

oxide,17

and

3


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layer-by-layer-assembled polymers18) were primarily used as the outside layer of microrockets. Unfortunately, their loading capacity and biodegradability in vivo and in other environments are still unsatisfactory.19 The lack of different types and quantities of side-chain functional groups also made them difficult to load enough kinds and numbers of payloads on their surface. Accordingly, it is critical to explore more biological materials to design the nontoxic microrocket with a higher loading capacity. Macromolecular polyamino acids (PAA) have been used in many biomedical fields,20 such as controlled drug delivery and release,21, 22 gene delivery,23 tissue

engineering,24

and

regenerative

medicine,25 because

of

their

good

biodegradability, biocompatibility and the physical and chemical characteristics of various side-chain functional groups (e.g., amino, carboxyl, sulfhydryl, and hydroxyl). 26, 27

However, the applications of PAA as a biomaterial in solution or in hydrogel

have been limited due to their complexity by chemical synthesis and poor mechanical properties.26 Moreover, the smooth and compact PAA membrane can be easily electropolymerized on the surface of an electrode to prepare chemically modified electrodes and biosensors.28,

29

Therefore, PAA, which is fabricated through

electropolymerization, is quite promising for making drug-delivery microrockets with excellent biocompatibility and biodegradability. In this study, we develop an effective drug-loading microrocket design using PAA. The microrocket consists of an outer polyaspartic acid (PASP) layer, a thin iron (Fe) intermediate layer, and a core of zinc particles (Zn). The iron layer is introduced in the tubular microrocket to facilitate magnetic control. Fe has also been widely used 4


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as a biomedical material due to its biocompatibility and high safe dosage.13 The Zn particles are electrodeposited into PASP/Fe to powerfully propel the microrockets in an acid environment by producing hydrogen bubbles

12

and to lower the oxidation of

Fe in the air. Doxorubicin (DOX), which is a chemotherapy medication used to treat cancer, could be easily bonded on the surface of bubble-propelled microrocket through the electrostatic interaction between the carboxyl groups of PASP and the amino groups of DOX in neutral solution. The DOX-loaded microrockets can be propelled using gastric acid as fuel, and they can be magnetically located, be trapped in the gastric mucus gel layer, and slowly release concentrated DOX payloads onto the stomach wall in acidic environment. The in vitro magnetic driving experiments demonstrated that a single DOX/PASP/Fe-Zn MR can reach and be trapped in the gastric mucus gel layer. The properties and functions of the synthesized DOX/PASP/Fe-Zn MRs were evaluated in a mouse model. The in vivo results illustrate

that the microrockets can permeate the gastric mucus gel layer and

increase the retention of the microrockets in the stomach without inducing an obvious toxic reaction. Furthermore, all materials in microrockets, including Fe, Zn and PASP microtubes, could be decomposed by the gastric acid or protease in the digestive tract.30 Therefore, PASP/Fe-Zn MRs would leave no harmful residue in vivo. Overall, using PAA as the surface material of the microrocket results in diverse functionalities and properties that extend the scope of practical biomedical applications of artificial micro/nanomotors.

5


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RESULTS AND DISCUSSION The cylinder-shaped drug-loading microrocket with an outer DOX/PASP layer, a thin Fe intermediate layer, and a core of Zn was fabricated using template-assisted electrodeposition and electrostatic interaction, as illustrated in Fig. 1A(a). The galvanic corrosion of Zn promoted by Fe makes the microrocket navigate in gastric acid by generating hydrogen bubbles. These drug-loading microrockets can be trapped in the gastric mucus gel layer and localize and concentrate the DOX payloads to the stomach wall, as shown in Fig. 1A(b). The targeted drug delivery is expected to be achieved by placing a strong magnet near a particular position on the stomach. The surface morphology and composition of DOX/PASP/Fe-Zn MR were confirmed by SEM and EDX in Fig. 1B. The SEM image reveals a smooth, compact, and intact PASP layer, which demonstrates that PASP was suitable to be used as the outside membrane material of microrockets. The PAA was formed by the polycondensation of carboxyl groups and amino groups between amino acid molecules through electropolymerization.31 Because L-ASP has two carboxyl groups, two kinds of possible PASP structures would coexist on the surface of microrockets, as illustrated in Equation 1. The EDX mapping analysis confirms the presence of C, N, Zn and Fe on or in the tube. Similar to the galvanic corrosion of other active metals, Zn first boosts proton depletion, reacts with acid, produces lots of hydrogen bubbles and propels the navigation of microrockets in acidic environments.13 Fe is used as the magnetic control component and the accelerator of the galvanic corrosion of Zn. Furthermore, because there are various kinds of side-chain functional groups (e.g., 6


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amino, carboxyl, and hydroxyl) on PASP, the PASP/Fe-Zn MRs can serve as drug-microcarriers via various intermolecular interactions. DOX, which is a common chemotherapeutic for treating a wide range of cancers,32 is adsorbed on the surface of a microrocket through a one-step process for its positive charge. Therefore, the adsorption is from the electrostatic interaction between the carboxyl groups of PASP and the amino groups of DOX.21,

33

The zeta potential analysis also shows that the

PASP/Fe-Zn MRs have a negative charge (-30.5 mV), whereas DOX/PASP/Fe-Zn MRs have a positive charge (2.65 mV), as illustrated in Fig 1S. In our work, the real medicine is loaded in bubble-propelled microrockets using the biological material PAA through electrostatic interaction instead of gold nanoparticles,12 organic dye from physical mixing,9 or SiO2 particles through vacuum infiltration.34 The EDX mapping analysis also confirms the presence of a few Cl on the surface of the microtube. These ions might have occurred because DOX-HCl is used in our experiment, and there may be a weak interaction between DOX and Cl- since DOX/PASP/Fe-Zn MRs have a positive charge (2.65 mV). H 2C

n HC

COOH NH2

electropolymerization

C

NH

HC X

CH2

COOH

C

NH

O

Y

COOH

L-天冬氨酸

体聚天冬氨酸

-PASP

L-ASP

amplified

HC CH2

COOH

The

O

SEM

images

and

confocal

(1)

体聚天冬氨酸

-PASP

fluorescence

images

of

DOX/PASP/Fe-Zn MRs before and after the metal-acid reaction in gastric acid simulant solution (pH = 1.2) are shown in Fig. 1C, which illustrates the completely 7


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full and empty tubular structures, respectively. Although almost all of the tube space is filled with Zn and Fe, as illustrated in Fig. 1C (a, c), the bubbles can still easily nucleate on the Zn core in the presence of acid due to the roughness of the filler. The microrocket was full of Zn and Fe in this study, which led to longer lifetimes compared to the previous hollow Zn-based MRs12 because of the prolonged reaction. The intermediate Fe layer also reacts with acid after a longer period of time. Fig. 1C (b, d) represents an almost empty tube structure that shows Zn and Fe in MR are completely consumed after a reaction with HCl. The MR loses its magnetic properties at the same time. Additionally, the PASP layer with 0.5-1 μm in thickness still keeps intact and smooth after the metal-acid reaction. The dissolving experiments show that PASP tubes cannot be destroyed with a typical organic solvent (methylene chloride, isopropanol, ethanol, acetone), an acid solution (0.5 M H2SO4, 1 M HCl) and a base solution (1 M NaOH) in 30 min. Therefore, PASP will be a robust surface material for fabricating MRs. Furthermore, because DOX had its own fluorescence characteristics (λex = 469 nm), confocal fluorescence images also showed the presence of DOX on the surface of the microtubes and thus confirmed loading of the DOX payload, as shown in Fig. 1C (c, d). The efficient and fast self-propulsion of MRs in the gastric acid simulant is demonstrated in Fig. 2 (and Videos S1 S2 and S3 in Supporting Information). The movement of PASP/Fe-Zn MRs is directionless when there is no other interference. They navigate along various trajectories, such as circles, lines or spirals, as illustrated in Fig. 2A (a, b) (Video S1, Supporting Information). The microrockets release 8


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numerous hydrogen bubbles when they are immersed into a gastric acid simulant according to the reaction in Equation (2): Zn (s) + 2H+ (aq)

Zn2+ (aq) + H2 (g)

9


(2)

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A a

Page 10 of 32

b

B

Zn

C

N

Fe

Cl

C

a

b

c

d

Figure 1. (A) The schematic structure of a DOX/PASP/Fe-Zn MR (a) and its application for effective and localization in the stomach (b). (B) SEM image of a full DOX/PASP/Fe-Zn MR and the EDX mappings of C, N, Zn, Fe and Cl inside the microrocket. Scale bar: 5 μm. (C) The SEM images and confocal fluorescence images 10


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of DOX/PASP/Fe-Zn MRs before (a, c) and after (b, d) the metal-acid reaction. Scale bars: 2 μm (a, b) and 5 μm (c, d).

The propulsion performance of microrockets is monitored and their moving trajectories are tracked and drawn in Fig. 2. The released bubbles produce a recoil force on the PASP/Fe-Zn MRs, which propel themselves forward at an average speed of 34.0 ± 7.2 μm/s (Fig. 2S(A)). As the reaction proceeds, the metal, including Zn and Fe, is continuously eroded and the MRs eventually stop. For the active metal-based MRs, their navigation lifetime is crucial and could restrict their practical applications. The previous Zn-based microrockets have shorter lifetimes (< 1 min in most cases)13, 16

due to their hollow tubular structure and high Zn activity with a very lower standard

redox potential (-0.76 V). Iron, instead of gold13,

16, 35,

is used to promote galvanic

corrosion due to the more negative standard redox potential (-0.44 V, compared to 0.15 V for Au) demonstrated in our work. Thus, the potential difference between two metals in MRs decreases from 0.91 V (Au-Zn) to 0.32 V (Fe-Zn). Therefore, it could prolong the navigation lifetime of PASP/Fe-Zn MRs (approximately 135 ± 37 s in gastric acid simulant). When MRs are in a mouse’s stomach, the pH of the gastric liquid would increase and then the lifetimes of these MRs would last longer in vivo compared to in vitro. In addition, because of the presence of Fe in microrockets, their motion could be directed using an additional magnetic field. The image (Fig. 2B (a)), trajectory image (Fig. 2B (b)) and corresponding Video S3 (in Supporting Information) show the magnetically guided motion of a PASP/Fe-Zn MR in the gastric acid 11


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simulant. The PASP/Fe-Zn MR could be magnetically directed in real-time to induce prospective tracks, such as two 90º turns at a speed of 29.2 ± 7.9 μm/s (Fig. 2S (B)). Compared to the autonomous movement, the propulsion speed of magnetic-controlled MRs was slower than self-propelled MRs. This result suggests that magnetic control changes the direction of PASP/Fe-Zn MRs, and also slows down the speed. Although it was reported that the surface functionalization would reduce the speed of Pt-based microrockets in the presence of 1–5% of the peroxide fuel,36 the speed of DOX/PASP/Fe-Zn MRs (31.8 ± 7.8 μm/s, in Fig. 2S(C)) was not affected by bonding with DOX. Such efficient propulsion of these MRs in the gastric acid simulant indicates considerable promise for in vivo evaluation and operation.

A

a

b

B

a

b

C

a

b

12


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Figure 2. (A) Self-propulsion of PASP/Fe-Zn MRs; (B) magnetic navigation of PASP/Fe-Zn MRs; (C) self-propulsion of DOX/PASP/Fe-Zn MRs suspended in the gastric acid simulant (pH = 1.2) with 1% SDS as surfactant, which correspond to Videos S1-S3, respectively. The images (a), trajectory images (b) of MRs are shown, respectively. The yellow trajectories indicate the motion. Scale bar: 30 μm.

Several controllable and tunable drug-release methods have been demonstrated in the literature, including

pH,37 temperature,38 electric fields,39 and light40

change-initiated release. The drug-loading capacity and the speed of drug release are crucial to the effect of therapy. To evaluate the drug-loading capacity of microrockets, the UV spectroscopies of DOX aqueous solution before adsorption and after adsorption by PASP/Fe-Zn MRs were studied as indicated in Fig. 3A. The loading capacity of DOX (DLC) is calculated to be 0.074 mg of DOX per 1 mg of PASP/Fe-Zn MRs from detecting the diversification of the absorbency value at 480 nm (maximum absorption wavelength of DOX). This outcome proves that PASP/Fe-Zn MRs have good loading capacity for drug delivery in neutral solution. When DOX/PASP/Fe-Zn MRs are added into a phosphate buffered solution (PBS, pH 7.4) for 20 minutes, the fixed rate of DOX (DFR) is approximately 98% according to the statistical calculation (Fig. 2S), suggesting that the release of DOX is very slow in neutral solution. However, it was reported that the pK0 for the PASP carboxylic groups is approximately 4.93,41 which was expected to cause the protonation of the carboxyl groups in the gastric acid (pH~1.2) and then cause the desorption of DOX 13


ACS Nano

from drug carriers. Under our experimental conditions, just a small amount of DOX is released into the gastric acid simulant after adding DOX/PASP/Fe-Zn MRs for 20 minutes, as shown in Fig. 3B. The DFR is approximately 88%, suggesting that although the release of DOX from microrockets in the gastric acid simulant is faster than that in the neutral solution, it is still a slower process. Therefore, after microrockets insert into the gastric mucus gel layer, targeted and sustained release of DOX proceeds.

A

B

0.4

0.03 Abs

0.2 0.1 0.0 400

0.05 0.04

0.3

Abs

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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0.02 0.01

450

500

550

0.00 400

600

450

500

550

600

Wavelength(nm)

Wavelength(nm)

Figure 3 (A) The UV emission curves of 0.1 mg/mL of DOX aqueous solution (red straight line) and the supernatant after adsorption by PASP/Fe-Zn MRs for 6 h (black dash line) in aqueous solution. (B) The UV emission curves in the gastric acid simulant (black dash line) and in the supernatant of gastric acid after adding DOX/PASP/Fe-Zn MRs for 20 minutes (red straight line).

To demonstrate the advantages of DOX/PASP/Fe-Zn MRs for potential biomedical applications, these microrockets were used to carry drugs and permeate 14


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the gastric mucus gel layer under the control of magnets in vitro. In Fig. 4A (Video S4, Supporting Information), a single DOX/PASP/Fe-Zn MR turns around without external interference, and it was difficult to arrive at the tissue slice. Its movement was changed from its original circular path to a straight line by a magnet (Fig. 4B). However, the microrocket could still not be directed to the tissue slice. After two attempts to prompt a U-turn with the magnet (shown in Fig. 4C-F), the microrocket finally arrives at the mouse stomach tissue slice and permeates it (Fig. 4G-H). These results demonstrate the significance of the microrocket propulsion and magnetic control to increase the delivery efficiency compared to self-propulsion. Therefore, the coefficient of the self-propulsion and the magnetic control would localize and concentrate the DOX payloads onto the stomach wall to promote targeted drug-delivery in vivo. A

B

C

D

E

F

G

H

Figure 4 Magnetic guidance of a single DOX/PASP/Fe-Zn MR in vitro. The stomach tissue slice is placed on the right side. Scale bar for (A-H):100 μm.

To study the in vivo delivery and payload release of DOX, mice were used as a 15


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model by orally administrating ultrapure water, DOX/PASP/Pt MRs and DOX/PASP/Fe-Zn MRs solution, respectively. After 30 minutes, the mice were euthanized, and their entire stomachs were excised. Rinsed with PBS, the whole stomachs were rinsed with PBS and analyzed with a fluorescence imaging technique. As illustrated in Fig. 5, because DOX/PASP/Pt MRs are not able to move by self-propulsion, the DOX/PASP/Pt MRs group, as well as the ultrapure water control group, display a small detectable fluorescence signal in the stomach, reflecting the self-fluorescence of possible food residue. The stomachs from mice raised with DOX/PASP/Fe-Zn MRs showed the strongest fluorescence intensity, which reflects substantial drug retention in the stomach under the same experimental conditions and with the same coatings. Comparing Fig. 5b and 5c, it seems that bubble propulsion plays a role for enhanced retention of the drug. It has been shown that there are many entangled and cross-linked mucin fibers on the surface of the stomach, which is covered with a 170-μm-thick gel-like mucus layer.12 When the Zn-loaded microrockets are actively propelled in the stomach, they will collide with the porous, slimy mucus layer and can be trapped in the gastric mucus gel layer, which leads to enhanced local drug retention. Our observations are similar to earlier studies in which the Zn-based microrockets were propelled in stomachs12 and the Mg-based microrockets were in the gastrointestinal tract, respectively.9 Consequently, the propulsion of the DOX/PASP/Fe-Zn MRs in the acidic stomach environment greatly enhanced their penetration and retention for DOX release. Otherwise, comparing with in vivo therapeutic Mg/TiO2 micromotors with antibiotic drug layer application for 16


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active drug delivery to treat gastric bacterial infection in a mouse model using clarithromycin as a model antibiotic and Helicobacter pylori infection as a model disease

a

b

c

Figure 5. Superimposed fluorescent images of the whole stomachs of mice collected 30 min post-administration of ultrapure water (a), DOX/PASP/Pt MRs (b), and DOX/PASP/Fe-Zn MRs (c), respectively. Scale bar: 5 mm.

In early studies, gold was always used as the substance for promoting galvanic corrosion in the design of microrockets due to its chemical inertness.12,

13

If these

microrockets are applied in vivo, gold will be left and may influence the functions of the organism. However, the inner Zn and Fe of our designed microrockets can be thoroughly consumed in the acidic stomach environment, and the polyaspartic acid layer can be finally consumed in the digestive tract due to its amino acid matrix. Moreover, Zn and Fe are biocompatible “green” nutrient trace elements that are vital for numerous body functions and processes. Therefore, all their metabolites are harmless, which suggests these microrockets would be a favorable micromotor to use as a drug carrier. Since toxicity is an important factor in any live animal experiment, in vivo applications of microrockets to evaluate the gastric toxicity of the administered 17


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microrockets in healthy mice. Mice are orally administered with the PASP/Fe-Zn microrockets solution and the same volume of water was used a control. The animals were monitored for general toxicity signs every 2 h over the first 10 h, and every 6 h for the later 6 days post-administration. No observable symptoms of pain, such as a hunched posture, lethargy, or unkempt fur were observed in the two groups. After 24 h, the mice were sacrificed, and sections of the animals’ stomachs were processed and stained to evaluate the toxicity of microrockets through histological analysis. The tissue sections are stained with hematoxylin and eosin (H&E), as illustrated in Fig. 6a and 6b. The gastric tissues had intact glandular mucosa with no differences in crypt and villi length, as well as number in between the microrockets-treated and water-treated groups. In addition, lymphocytic infiltration into the mucosa and submucosa was not apparent, which suggested there were no signs of gastric inflammation. The potential toxicity of the PASP/Fe-Zn MRs was further evaluated using gastric tissue sections in a terminal deoxynucleotidyl transferase-mediated deoxyuridine TUNEL assay to examine the level of gastric epithelial apoptosis as an indicator of gastric mucosal homeostasis, which is shown in Fig. 6c and 6d. There was no apparent increase in gastric epithelial apoptosis for the microrockets-treated groups compared to the water control groups. The in vivo toxicity studies indicate that the orally administered MRs are safe for mouse models. Therefore, PASP/Fe-Zn MRs will achieve a zero-waste profile in vivo and they are nontoxic, biocompatible and biodegradable. Overall, such PAA-based microrocket results provide diverse functions and properties and extends the practical applications of artificial 18


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micro/nanomotors.

a

b

c

d

Figure 6. Toxicity evaluation of PASP/Fe-Zn MRs. The mouse stomachs were treated with PASP/Fe-Zn MRs, and the same volume of water was used as a control. After 24 h, the mice were sacrificed and sections of the mouse stomachs were processed and stained in a H&E assay (a, b) and TUNEL assay (c, d). Scale bars: 250 μm.

CONCLUSIONS We have demonstrated that attractive polyamino-acid based microrockets can potentially be used as ‘zero-waste’ micromachines for active and targeted delivery of an anticancer drug in vivo. In particular, there are various kinds of side-chain functional groups (e.g., amino, carboxyl, sulfhydryl, and hydroxyl) on polyamino acids, and the PASP/Fe-Zn MRs can serve as microcarriers via various intermolecular interactions for diverse bioapplications in vitro and in vivo. The motor’s zinc core reacts with the gastric fluid to generate a propulsion force associated with the proton 19


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depletion reaction. The propulsion of the DOX/PASP/Fe-Zn MRs in the acidic stomach environment greatly improves their penetration and enhanced DOX retention. The PASP polymer and Zn, as well as Fe, leave no harmful products following their movement, cargo delivery, and self-destruction in vivo, making these PAA-based microrockets attractive for various biomedical applications.

METHODS Fabrication of PASP/Fe-Zn MRs. The PASP/Fe-Zn and PASP/Pt MRs were prepared by electrodeposition using cyclopore polycarbonate membranes (5 μm diameter, 7060-2513; Whatman, Maidstone, UK) as the templates. The membrane was assembled in a plating bath with the 75-nm-thick gold film side facing down to serve as a working electrode and put in contact with a stainless-steel sheet. A Pt wire and an Ag/AgCl electrode (with 3 M KCl) were used as counter and reference electrodes, respectively. First, the PASP layer of the microrocket was prepared by electropolymerization (A CHI 832B electrochemical workstation) with cyclic voltammetry (-0.6 V2.0 V, 12 cycles, 20 mV/s) in 5 mM of aspartic acid aqueous solution containing 0.1 M citric acid and sodium dihydrogen phosphate buffer (pH = 6). Second, a Fe layer was prepared with a potentiostatic method (potential: -1.0 V; charge: 2.0 C) from a Fe plating solution containing 260 g/L FeSO4 and 20 g/L H3BO3 (adjusted to pH = 2.5 by H2SO4). Nitrogen (N2) was bubbled before and during the Fe electrodeposition to prevent the oxidation of Fe(II) to Fe(III). Finally, a zinc layer was electrodeposited at -1.2 V with a charge of 10.0 C from a zinc plating solution 20


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containing 80 g/L ZnSO4 and 20 g/L H3BO3 (adjusted to pH = 2.5 by H2SO4). As a passive control, microrockets with a Pt inner layer rather than a Zn inner layer were prepared by electrodeposition at -0.28 V with a charge of 1.0 C in 1 g/L chloroplatinic acid solution. Then, the overgrown metal and the Au film on the two sides of the membrane were polished completely with 34 μm aluminum oxide, which was dispersed by isopropyl alcohol to prevent the oxidation of iron. The cleaned membrane was dissolved in methylene chloride for 30 min to completely release PASP/Fe-Zn MRs. Then, the solution was centrifuged at 5000 rpm for 1 min to collect microrockets, and the procedure was repeated once. These microrockets were washed with isopropanol and ethanol twice. Before use, the microrockets (approximately 5 mg per membrane) were washed with ultrapure water twice and 0.3 mL of ultrapure water were added to disperse them. The structure and composition of the microrockets were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX, SU8010, Hitachi, Japan). Movement and magnetic driving in vitro. All the movement experiments were performed in gastric acid simulant (pH = 1.2) with 1% SDS as surfactant. In each test, a drop of solution containing PASP/Fe-Zn MRs or DOX/PASP/Fe-Zn MRs were dispersed on a glass slide. The movement direction of the microrockets was controlled by changing the orientation of an external magnet. The microscopy images and the videos of microrockets in motion were recorded with optical microscopy (TCS SP8, Leica Microsystems, Inc. Germany). The speed and tracking of microrockets was calculated from the recorded videos and analyzed using Video Spot Tracker software. 21


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Loading DOX onto microrockets. To study the drug-loading capacity of microrockets, DOX was selected as a drug payload that could be carried by the PASP/Fe-Zn MRs. The PASP/Fe-Zn MRs or PASP/Pt MRs collected from a membrane (in 50 μL of ultrapure water) were added to 1.0 mL of 0.1 mg/mL DOX aqueous solution, and the mixture was incubated for at least 6 h in darkness to achieve the largest adsorption of DOX. Then, the solution was centrifuged at 5000 rpm for 3 min, and the microrockets were washed twice with ultrapure water. The supernatant with free DOX was collected, and the free DOX concentration (WDOX-free) was determined by UV-Vis spectroscopy at 480 nm with a UV-vis Spectrometer (UV-2600, Shimadzu, Japan).22 Therefore, the weight of DOX loading on PASP/Fe-Zn MRs (WDOX-MRs) was calculated using the formula (1): WDOX-MRs = WDOX-total - WDOX-free

(1)

Where WDOX-total = total weight of DOX added in the microrockets solution. The loading capacity of DOX (DLC) on PASP/Fe-Zn MRs was calculated using the formula (2): DLC (w/w) = WDOX-MRs/Wmicrorockets

(2)

Where Wmicrorockets = weight of PASP microrockets in a membrane. Because some of the DOX would be released in an acidic environment,42 the fixed rate of DOX (DFR) on PASP/Fe-Zn MRs in the gastric acid simulant was calculated using the formula (3): DFR (%w/w) = (W DOX-MRs - W DOX-acid) / W DOX×100

22


(3)

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where W DOX-acid = weight of DOX in the gastric acid supernatant after 20 minutes of incubation. Zeta Potential Study. PASP/Fe-Zn MR as well as DOX/PASP/Fe-Zn MR (0.3 mg) were dispersed in 1 mL of ultrapure water, respectively. Then, the zeta potential was characterized by determining the electrophoretic mobility using a Zetasizer Nano ZS (Malvern Instrument, UK) at room temperature. Each sample was measured six times to get the mean value. In Vivo Release Study. ICR male mice at six-weeks-old, which were purchased from Shandong University Experimental Animal Center, were fasted for 12 h. Then, nine mice were randomly divided into three groups (n = 3). They were orally administered with 0.3 mL of the DOX/PASP/Fe-Zn MRs solution (acquired from a half membrane) as an experimental group, or 0.3 mL of the PASP/Pt MRs loaded with DOX (DOX/PASP/Pt MRs) solution as the control group. The third group was given the same volume of ultrapure water as a blank group. The mice were sacrificed 30 min post-administration, and their stomachs were cut open along the greater curvature. The gastric contents were removed, and the gastric fluid containing unanchored microrockets was washed away. Subsequently, the stomachs were imaged using an intelligent visual inspection system (IVIS Lumina XRMS Series III, PerkinElmer, America). In Vivo Toxicity Study. To evaluate the acute toxicity of PASP/Fe-Zn MRs in vivo, six-week-old ICR male mice were orally administered with 0.3 mL of PASP/Fe-Zn MRs solution. Mice treated with ultrapure water were used as controls. 23


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After 24 hours, the mice were sacrificed, and their stomachs were removed for histological analysis. The general toxicity signs were monitored for every 2 h over the first 10 h and every 6 h for the later 6 days post-administration. The longitudinal sections of gastric tissue were fixed in neutral-buffered with 4% (v/v) formalin for 12 h then transferred into a 30% sucrose solution. The tissue sections were cut at a 5-μm thickness and stained using a hematoxylin and eosin (H&E) assay, as well as a triphosphate nick-end labeling (TUNEL) assay.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website Supporting Figures 3 (PDF) Video 1 (AVI) Video 2 (AVI) Video 3 (AVI) Video 4 (AVI)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] Author Contributions 24


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†M. Z., and T. H. contributed equally to this work.

ACKNOWLEDGMENTS This work was financially supported by Natural Science Foundation of Shandong Province (No. 2014ZRB01437); Project of Shandong Province Higher Educational Science and Technology Program (No. J14LC03); Shandong Graduate Student Novel Item (No. SDYY13012).

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ToC graphic

A microdevice that consists of a polyaspartic acid (PASP) microtube, a thin Fe intermediate layer, and a core of Zn can propel itself using gastric acid as a fuel. After adsorbing doxorubicin onto the PASP surface, the microrocket can carry a drug, be magnetically located, insert into the gastric mucus gel layer, and increase drug retention in the stomach without inducing obvious toxic reactions.

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Iron

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