Real-Time Fluorescence Tracking of Protoporphyrin Incorporated

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Real-time fluorescence tracking of protoporphyrin incorporated thermosensitive hydrogel and its drug release in vivo Xia Dong, Chang Wei, Tianjun Liu, Feng Lv, and Zhiyong Qian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11493 • Publication Date (Web): 05 Feb 2016 Downloaded from http://pubs.acs.org on February 8, 2016

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ACS Applied Materials & Interfaces

Real-time Fluorescence Tracking of Protoporphyrin Incorporated Thermosensitive Hydrogel and Its Drug Release in Vivo

Xia Dong1, Chang Wei1, Tianjun Liu1 , Feng Lv1*, Zhiyong Qian2* 1 Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, PR China 2 State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, P.R. China

*Corresponding author: Feng Lv Institute of Biomedical Engineering Chinese Academy of Medical Sciences & Peking Union Medical College Tianjin 300192, PR China Tel/Fax: 86-22-87893236, E-mail: [email protected] (Lv F) Zhiyong Qian State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, P.R. China Tel/Fax: 86-28-85501986, E-mail: [email protected] (Qian ZY).

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ABSTRACT

Fluorescence imaging in vivo will pave an important way for the evaluation of biomaterials. The major advantage of fluorescence imaging compared to other imaging modalities is the possibility of tracking two or more fluorescence probes simultaneously with multispectral fluorescence imaging. It is essential to elucidate the location, erosion, drug release and resection of implanted biomaterials in vivo. Herein, a thermosensitive hydrogel with a protoporphyrin core based on a PEG and PCL copolymer (PCL-PEG-PPOR-PEG-PCL) was synthesized by ring-opening polymerization using protoporphyrin as a fluorescence tag. The optical properties of the hydrogel were investigated by UV-vis and fluorescence spectroscopy in vitro and by fluorescence imaging system in vivo. The hydrogel erosion and drug delivery in vivo were monitored and tracked by multispectral fluorescence imaging system in nude mice. The results show that the thermosensitive hydrogel exhibits fluorescence and injectability in vivo with good biocompatibility. Through the modality of fluorescence imaging, the status of the hydrogel is reflected in situ in vivo including its location and erosion. Multispectral analysis separates the autofluorescence signals from the specific label and provides the ability to locate the drug and carrier. The protoporphyrin incorporated thermosensitive hydrogel can be a potential visiable biomedical implant for tissue repair or drug delivery.

Keywords: Protoporphyrin, Thermosensitive hydrogel, Fluorescence imaging, Drug release, Multispectral fluorescence

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1. Introduction In situ imaging technologies are a strategic priority in research on biomaterials on account of their real-time, non-destructive, longitudinal, quantitative and three-dimensional analysis 1. Histological and immunohistochemical assays do not provid an accurate volume assessment containing information on the functional status in situ because the samples must be evaluated ex vivo. There is a great need for the development of imaging tools to implement the monitoring and assessment of biomaterials. Traditional imaging modalities have been considered for tracking or monitoring the implanted biomaterials in vivo including ultrasonography (US), computed tomography (CT), SPECT and magnetic resonance imaging (MRI)

2-4

. Although these medical

imaging modalities play an important role in the evaluation of biomaterials in vivo, fluorescence imaging has been shown to be an advanced tool for tracking the in vivo fate of biomaterials, drugs and cells due to its easy setup, low cost, high sensitivity, low-energy radiation, non-invasion and long-term observation 1,5. Fluorescent polymer implants show significant functionality and special advantages for tissue engineering materials or drug delivery carriers.

In vivo fluorescence

6,7

, biomaterial-associated

imaging has been applied to the long-term monitoring of glucose inflammation monitoring drug release

11,12

8,9

, bone tissue engineering regrowth

10

, and materials degradation and

with fluorescence biomaterials. A major advantage of fluorescence imaging

compared to other imaging modalities is the possibility of tracking two or more fluorescent probes simultaneously with multispectral fluorescence imaging

13,14

. Multispectral fluorescence

imaging has been serviced for monitoring the biofate of biomaterials and tracking drug delivery. For example, Hoffmann et al prepared dual fluorescent HPMA copolymers for passive tumor targeting with pH-sensitive drug release in order to track the drug and carrier in vivo simultaneously 12. They observed the body fate of the polymer and the model drug noninvasively by dual fluorescent labels. Multispectral fluorescence imaging can provide the location and status of the drug and carrier clearly. This advanced method is also important for implanted biomaterials to track their erosion and drug delivery. Fluorescence tagging is a key component of fluorescent implant and needs to be further developed.

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Porphyrins have played a significant role in the biological, photo-physical and catalytic fields owing to the special optical properties from their extended π-conjugated electronic macrocycle structures

15-17

. Based on their large visible and near infrared absorption, molar extinction

coefficient, acceptable fluorescent quantum yield and excellent biocompatibility, porphyrins and their derivatives have been widely considered in biomedical fields as new-generation photosensitizers, fluorescence imaging probes and oxygen carriers

18-20

. Fluorescence imaging

with porphyrin has also attracted our interest and several porphyrin-based fluorescent probes were investigated for in vivo imaging

21,22

. The fluorescence imaging agents that are approved for

clinical use by the FDA are indocyanine green and methylene blue, which show many advantages for fluorescence labeling and imaging23-25. However, their wide applications are seriously limited by their drawbacks including complex synthesis, high cost and low quantum efficiency. Porphyrins are superior fluorescent tags for in vivo imaging because they have a large molar extinction coefficient, acceptable fluorescent quantum yield and excellent biocompatibility. Protoporphyrin is a special porphyrin compound that plays a key physiological function as a component of hemoglobin, which assigns it favorable biocompatibility exceeding that of other synthetic porphyrin compounds. Protoporphyrin conjugated compounds tend to be ideal oxygen carriers and photodynamic therapy agents

20,26

. Nevertheless, the decrease in the effectiveness of

the photo-dynamic effect seriously limits their applications because of the aggregation from π-stacking and hydrophobic interactions in aqueous media, even if protoporphyrin compounds have the advantages of water solubility and favorable biocompatibility. Regretably, protoporphyrin is hardly applied to fluorescence imaging as a probe or a tag due to its faint fluorescence from molecular aggregation in water solutions. So far there have been no related reports regarding to protoporphyrin incorporated biomaterials for fluorescence imaging. The superordinary in vivo imaging enables providing an extensive perspective of porphyrin incorporated hydrogels27,28. It will be a great challenge to develop fluorescent biomaterials with protoporphyrin as a tag.

As representative implanted biomaterials, thermosensitive synthetic hydrogels are injectable hydrogels with hydrophilic polymer networks retaining a large amount of water. They can be administered under mild conditions and then form a non-fluid gel with the change in temperature. 4


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They are widely applied in soft tissue engineering, drug delivery, surgical implants and so on because of the advantages that rely on their high moldability, minimally invasive delivery and effective drug encapsulation

29-31

. Poly(ethylene glycol)(PEG) and poly(ε-caprolactone)(PCL) are

both well-known FDA-approved biodegradable materials that are the constitutive units of thermosensitive hydrogels

32,33

. PEG-PCL hydrogels do not produce an acute acid environment

that leads to an obvious inflammatory reaction. Thermosensitive hydrogels based on PEG and PCL possess various applications in the biomedical and pharmaceutical fields, including drug delivery, tissue repair and surgical implants in view of their injectability

34,35

. To elucidate the

status of an implanted hydrogel in vivo, the location, erosion and drug release of the hydrogel are essential and crucial to monitor and track by multispectral fluorescence imaging.

In this article, a thermosensitive hydrogel with a protoporphyrin core based on a PEG and PCL copolymer (PCL-PEG-PPOR-PEG-PCL) was synthesized by ring-opening polymerization using protoporphyrin as a core of the polymer backbone. The PCL-PEG-PPOR-PEG-PCL fluorescent hydrogel is designed with advantages of injectability and fluorescence ability. This can avoid the need for surgical implantation and removal of the hydrogel and generates an advanced method for conveniently monitoring implants in vivo. The thermosensitivity and optical behavior in vitro were investigated with tube-inverting testing, rheological analysis, differential scanning calorimetry, UV-visible and fluorescence spectrophotometry. Importantly, hydrogel erosion and drug delivery in vivo were monitored and tracked by a multispectral fluorescence imaging system with nude mice as models. The PCL-PEG-PPOR-PEG-PCL hydrogel can be a potential visiable biomedical implant for drug and gene delivery and tissue regeneration as well as for tissue adhesion prevention and wound covering with in vivo monitoring and non-invasive tracking.

2. Experimental Section 2.1 Materials

Protoporphyrin( ≥ 95 %, Aladdin) was provided by Shanghai Jingchun Biotech Corporation. Poly(ethylene glycol) (PEG, Mn = 1000, Merck) was vacuum-dried at 60 °C for 12 hours before use. ε-Caprolactone (ε-CL,Aladin) was purified by vacuum distillation. Stannous octoate

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(Sn(Oct)2,Aladin) and other reagents were all analytic reagent (AR) grade.

Nude mice (seven weeks old, 20–25 g) were used. All animal procedures were conducted following the protocol approved by the Institutional Laboratory Animal Ethics Committee, and all animal experiments were performed in compliance with the Guiding Principles for the Care and Use of Laboratory Animals, Peking Union Medical College, People’s Republic of China. Animals had free access to food and water. 2.2 Synthesis and characterization of PCL-PEG-PPOR-PEG-PCL copolymer

PCL-PEG-PPOR-PEG-PCL copolymer was synthesized by the ring-opening copolymerization of ε-CL initiated by protoporphyrin-conjugated PEG using stannous octoate as a catalyst according to Fig. 1. PEG 1000 (4 g) was reacted with protoporphyrin (40 mg) in DMF (20 mL) at room temperature for 24 hours under the catalysis of EDC and DMAP. The reacted solution was washed with water, precipitated with cool ether and dried under vacuum to give the crude protoporphyrin-conjugated PEG. Then, it was purified to remove the PEG molecule by dialysis. Next, protoporphyrin-conjugated PEG (1 g) and ε-CL (1 g) were polymerized under the catalysis of Sn(Oct)2 (0.1 g) in a polymerization tube under vacuum at 120 °C for 24 hours. The mixture was dissolved in dichloromethane and then precipitated with cold petroleum ether, filtrated and dried to provide the PCL-PEG-PPOR-PEG-PCL copolymer. 1

H-NMR spectra were recorded on a VARIAN INOVA instrument at 500 MHz using CDCl3 as

solvent and TMS as an internal reference. Gel permeation chromatography (GPC) on a Malvern TDA305 was used to determine the molecular weight and polydispersity of the copolymers. The samples were dissolved in THF to a concentration of 2 mg/mL.

2.3 Sol–Gel–Sol phase transition, rheological and thermal behavior

The ol–gel–sol phase transition behaviors of PCL-PEG-PPOR-PEG-PCL copolymers were observed using the tube-inversion method in a 4 mL tube at a concentration of 40 % with a heating rate of 1 °C min-1 from 10 °C to 60 °C. The gelation responsive time of the hydrogel at 37°C was measured using the tube-inversion method. The sol–gel–sol phase transition was determined by

6


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inverting the tube horizontally, and the conditions of gel and sol were defined as ‘‘no flow’’ and ‘‘flow’’ in 1 min, respectively. Rheological measurements of the sol-gel-sol transition of the hydrogel were carried out using a rheometer (AR 1000, TA Instruments, USA). The copolymer aqueous solution (40 wt. %) was placed between parallel plates of 40 mm diameter and with a gap of 52 μm. The heating rate was set as 2 °C min-1 over the range of 10-60 °C. The storage modulus (G’) and loss modulus (G’’) were measured under a frequency of 1 Hz and a rate of 2.0 rad s-1. Differential scanning calorimetry (DSC) (Q 2000, TA instruments, USA) was used to analyze the thermal properties of the copolymer in the temperature range from -10 to 80 °C under a nitrogen atmosphere at a heating and cooling rate of 5 °C min-1.

2.4 Optical measurement in vitro

UV-vis and fluorescence spectra of the PCL-PEG-PPOR-PEG-PCL copolymer were evaluated at a concentration of 2 mg mL-1 in water solution by a multimode microplate spectrum photometer (Varioskan TM Flash, ThermoFisher Scientific, USA). The fluorescence imaging of the hydrogel in vitro was taken by an in vivo imaging system (Maestro EX, CRI, USA).

2.5 Fluorescence tracking of degradation in vivo and organ distribution ex vivo

Nude mice were housed in cages with free access to food and water and were randomly assigned to the subcutaneous injection group and control group (n = 3 for each group). In the experimental group, 200 µL aqueous solution of PCL-PEG-PPOR-PEG-PCL copolymer was injected in the subcutaneous tissue of the back at a concentration of 40 %. The in vivo imaging was recorded after administration from 0 day to 21 days. At predetermined time points, the mice were anesthetized by an intraperitoneal injection of chloral hydrate at a concentration of 4 %. Imaging was taken using a CRI imaging system with an exposure time of 200 ms. The excitation wavelength was set at 595 nm, and the emission wavelength was chosen from 635 nm to 800 nm. At the end of the imaging, the anesthetized mice were sacrificed and imaging of the organs was performed to evaluate the distribution of fluorescent materials. The fluorescence images of the organs were analyzed using the CRI Analysis Software. After imaging, the organ tissues were immediately immersed in phosphate-buffered saline with 4 % formaldehyde at pH 7.4 and 4 °C

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for 24 hours. Then, they were stained with hematoxylin-eosin after immobilization and sectioning.

2.6 Morphology of hydrogel before injection and after erosion

Scanning electron microscopy (SEM) (S4800, Hitachi, Japan) was employed to observe the morphology of the hydrogel. Samples were taken before the injection and after the erosion of the hydrogel for 21 days. They were frozen in liquid nitrogen and lyophilized for 72 hours and then were observed after gold sputtering.

2.7 Drug release from hydrogel in vitro by absorbance spectrum and in vivo by multispectral fluorescence imaging A water soluble fluorescent dye, rhodamine, was chosen as a model drug. The in vitro drug-release profile of rhodamine from the hydrogel was studied in PBS at pH 7.4 and 37 °C. At preset time points, 2 mL of the extra fluid was taken for rhodamine analysis by absorbance spectrum and 2 mL of fresh phosphate buffer was added. The concentration of rhodamine was calculated from a standard curve of known rhodamine absorbance of 554 nm. All experiments were performed in triplicate.

Multispectral imaging was carried out to track the carrier and drug in the Maestro in vivo imaging system from CRI. The excitation wavelength was set at 595 nm, and the emission wavelength was chosen from 635 nm to 800 nm. The drug and carrier can be distinguished at the same excitation according to the difference in emission. The Maestro software was used to separate the spectral species from the cube file and overlay the single compounds with yellow and blue. The unmixed grayscale images of the single components from drug can be calculated quantitatively. The in vivo imaging was recorded to track the release of the drug for 5 days.

3 Results and Discussion

3.1 Preparation and structural characterization of copolymer

A protoporphyrin incorporated copolymer based on PEG and PCL was synthesized with a simple and convenient polymerization, as seen in Fig. 1. Based on the cross-linking of

8


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protoporphyrin and PEG, PCL segments were linked to the protoporphyrin conjugated PEG compound as hydrophobic units to ensure the thermosensitivity and fluorescence of the hydrogel. Amphiphilic copolymers with a porphyrin core based on PEG and PCL are always synthesized by ring-opening polymerization or its combination with atom transfer radical polymerization36-38. A porphyrin incorporated hydrogel with a four-arm PEG-PCL copolymer (POR-PEG-PCL) has been prepared by us by ring-opening polymerization using porphyrin-PEG as an initiator39,40. The PCL-PEG-PPOR-PEG-PCL copolymer is a linear polymer owing to the bifunctional group from protoporphyrin, whereas the POR-PEG-PCL copolymer exhibits a star-shaped structure because synthetic porphyrin carries four reactive groups. Porphyrin hydrogels can also be prepared by physical encapsulation or surface absorption and porphyrin tends to be released from the hydrogel as an active drug without enough stability on the backbone of the hydrogel41,42. As porphyrin cannot be cut off from chemically cross-linked porphyrin hydrogels due to their extraordinary stability, the PCL-PEG-PPOR-PEG-PCL copolymer can provide fluorescence tracking for a long period of time. Instead of providing ectogenic fluorescence labeling to the polymer, a fluorescent hydrogel copolymer was synthesized using fluorescent protoporphyrin as the backbone and core. Compared with other fluorescent dyes at the polymer end, the protoporphyrin core in the polymer backbone not only avoids the early selective breakage of the fluorescent tag in the ending, ensuring feasible fluorescence efficiency but also decreases the adverse effect to the biotissue of fluorescent dyes. Protoporphyrin incorporated copolymers exhibit intense fluorescence as a result of the linkage to a long chain polymer including PEG and PCL. As seen in the 1H-NMR spectrum of PCL-PEG-PPOR-PEG-PCL (Fig.S1), the characteristic peaks of the PCL unit are shown at 1.36,1.62,2.29 and 4.04 ppm, which are attributed to the methylene protons of –(CH2)3-,-OCCH2- and –CH2OOC-. The characteristic peak at 3.64 ppm is the signal of –CH2CHO- in the PEG. Besides, there are two characteristic minor peaks of protoporphyrin at 6.51 ppm and 8.05 ppm, signifying the vinyl Hb trans to porphyrin and the Ha trans to porphyrin. Other characteristic peaks cannot be observed because of their weak signals. All these signals confirm the successful synthesis of the PCL-PEG-PPOR-PEG-PCL copolymer. The mean molecular weight and molecular weight distribution (PDI, Mw/Mn) of the copolymer

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were determined by GPC (Fig.S2). It has a mean molecular weight Mn of 3771 and a PDI (Mw/Mn) of 1.362.

3.2 Sol–gel–sol phase transition, rheological and thermal behavior

A thermosensitive hydrogel can be injected at the target location as a sol, and then it forms a gel in the solid state due to the sol-gel phase transition at around physiological temperature. A PCL-PEG-PCL thermosensitive hydrogel has been successfully exploited by us 43,44. Based on its similar structure, the PCL-PEG-PPOR-PEG-PCL hydrogel has an ideal thermosensitivity as a matter of course. The bridge-link and central insert of protoporphyrin would not significantly hamper the thermosensitivity of the hydrogel. The sol-gel-sol phase transition of the PCL-PEG-PPOR-PEG-PCL hydrogel in vitro is shown in Fig. S3 as demonstrated by the tube-inversion method. Just like the PCL-PEG-PCL hydrogel, the PCL-PEG-PPOR-PEG-PCL hydrogel is an injectable flowing sol at room temperature while it forms a non-flowing gel at physiological temperature. The gelation response of the PCL-PEG-PPOR-PEG-PCL hydrogel takes only 1 min from solution to gel at 37°C using the tube-inversion method. The accurate location of the hydrogel can be achieved after injection and implantation with a rapid response. With the further increase of the temperature, the non-flowing gel transforms into a precipitate with the gel-sol phase transition. As the hydrogel cannot complete the gel-sol transition in vivo at approximately 37 ℃, it can retain the gel state until it degrades gradually at body temperature. It displays a lower critical gelation temperature and an upper critical gelation temperature with a special temperature-dependent sol-gel transition in water. A

rheological

study

further

confirms

the

thermal

response

behavior

of

the

PCL-PEG-PPOR-PEG-PCL hydrogel, as shown in Fig. S4A. The change in the elastic modulus (G’)

and

viscous

modulus

(G’’)

reflect

the

sol-gel-sol

transition

of

the

PCL-PEG-PPOR-PRG-PCL copolymer in the temperature range of 10-60 °C. When the temperature is less than 29 ℃, the copolymer has a low elastic modulus (G’) and viscous modulus (G’’), with the viscous modulus (G’’) only slightly higher. When the elastic modulus is equal to the viscous modulus, the lower critical gelation temperature (LCST) and sol-gel transition temperature are reached. This suggests that the copolymer is a flowable fluid with

10


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low viscosity below the LCST. The copolymer has an obvious thermal sensitivity, with an LCST of 29 ℃. With the further increase in temperature, the increase rate of elastic modulus is higher than that of the viscous modulus. The higher elastic modulus of the copolymer reveals that the hydrogel transforms to a solid gel from a flowable sol. The copolymer can not flow randomly in a gel state at the physiological temperature of approximately 37 ℃. When the temperature exceeds to 41 ℃, the rapid decrease of the elastic modulus and viscous modulus indicates that the hydrogel has changed to a flowable sol. The thermal properties of

the

PCL-PEG-PPOR-PEG-PCL copolymer are shown in Fig. S4B by DSC. Just like in other PEG-PCL copolymers, two endothermic transitions are displayed in the range of 30-50 ℃ in the heating process of PCL-PEG-PPOR-PEG-PCL. They are attributed to the melting of the PEG and PCL segments, respectively. The cooling process also shows two exothermic peaks in the range of 9-15 ℃, which are indication of the crystallization of PEG and PCL, respectively.. The intervention of protoporphyrin brings partials change to the thermal properties of the PCL-PEG-PPOR-PEG-PCL copolymer and slight differences in the sol-gel-sol transitions of the hydrogel 39.

3.3 Optical characterization of protoporphyrin incorporated copolymer and hydrogel in vitro

The absorption and fluorescence spectra of the PCL-PEG-PPOR-PEG-PCL copolymer in water solution are shown in Fig. 2. In the UV-vis spectrum of the PCL-PEG-PPOR-PEG-PCL copolymer, the typical absorption peaks of porphyrin compounds are displayed with an intense sharp Soret band at 420 nm and two weak wide Q-bands at 540 nm and 600 nm in the near-infrared region. The Q-band absorption is beneficial for in vivo imaging because the optical signal of the shorter wavelength is rapidly attenuated in biotissue. Two primary emission peaks are shown at 590 nm and 640 nm, with excitation wavelengths of 420 nm, 540 nm and 590 nm respectively. To provide the in vivo imaging with low autofluorescence and interference of the background, excitation and emission should occur with longer wavelengths, even though the fluorescence intensity is lower than that at 420 nm. Compared with protoporphyrin at the same concentration, the intense fluorescence of the PCL-PEG-PPOR-PEG-PCL copolymer indicates the

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non-aggregated state of the porphyrin molecules due to the extension of the polymer chains. The aqueous solution of protoporphyrin has little emission because of the self-quenching of its fluorescence, while fluorescence quenching can be avoided in the protoporphyrin polymer through the conjugation with a long chain polymer (Fig. S5A). This phenomenon has been confirmed in other water-soluble porphyrin compounds 21. The aggregation of porphyrin comes from π-stacking and hydrophobic interactions in aqueous media, leading to the resulting reduction in the effectiveness of the photo effect for fluorescence imaging. Here, the protoporphyrin conjugated macromolecule shows intense fluorescence signals because the high density of protoporphyrin is spatially

enforced

to

porphyrin-cross-linked

prevent

molecular

hydrogels

have

self-quenching. confirmed

this

Related

reports

phenomenon27,28.

on

other

Although

PCL-PEG-PPOR-PEG-PCL transforms to a solid gel through a sol-gel transition, it still maintains intense fluorescence as measured by the CRI imaging system in vitro . Moreover, multispectral fluorescence imaging separates the protoporphyrin incorporated hydrogel and protoporphyrin solution with red and blue according to their differences of emission, even if the fluorescence of the protoporphyrin solution is very faint. This demonstrates that hemoglobin with protoporphyrin will not generate any interference for the imaging of the hydrogel. This ensures the feasibility of noninvasive fluorescence tracking of the PCL-PEG-PPOR-PEG-PCL hydrogel in vivo.

3.4 Fluorescence tracking of degradation in vivo The degradation or erosion of hydrogels plays a significant role that determines the tissue repair period or drug release rate 45. Generally, in vitro or ex vivo assays have been applied to assess the degradation of biomaterials including the gravimetric or volume determination of periodic samples as well its physicochemical properties of molecular weight, mechanical properties, morphology and viscosity 46,47. However, in vitro and ex vivo assays cannot easily reveal the status of the implanted hydrogel in vivo due to the complex biotissue system and the intricate environmental forces. It is crucial to assess the material erosion and drug delivery in vivo. The in vivo degradation of materials can permit long-term, noninvasive fluorescence monitoring and image-guided surgical resection in vivo

28,48,49

. We also evaluated the fluorescence decay of a

thermosensitive porphyrin incorporated hydrogel with a four-arm PEG-PCL copolymer 39. Now, a novel PEG-PCL hydrogel with protoporphyrin core was investigated in detail to monitor the 12


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erosion and degradation. The linkage of protoporphyrin makes it visible in the location, tracking and resection of the hydrogel by fluorescence imaging. Fluorescence guiding can enable the hydrogel to be injected at the targeted site precisely and rapidly.

Whole animal fluorescence imaging was performed to monitor and track the implanted hydrogel in nude mice by subcutaneous injection in the back. Due to the visibility of the fluorescent hydrogel, it can be located in the targeted area and surgical resection can be performed using image- guiding in vivo. The fluorescence imaging was performed in the emission range from 630 nm to 800 nm at the excitation wavelength of 595 nm, and the intense fluorescence of the hydrogel can be observed (Fig. S5B). Compared with shorter excitation wavelengths, the hydrogel has obvious fluorescence along with a low background signal at the excitation wavelength of 595 nm. Generally, near infrared light is ideal for in vivo fluorescence imaging at the wavelengths from approximately 600 nm to 1000 nm. The depth penetration at short wavelengths is limited by the biological chromophore hemoglobin from the strong visible light absorption. Other biological components including water and lipids introduce weak interference from the background because while they are optically transparent from the visible to the near infrared region, they strongly absorb infrared light. Light scattering and autofluorescence from biological tissue are low in the near infrared region, including from elastin, collagen, and other biological fluorophores

50

.

Accordingly, the emission range from 630 nm to 800 nm at the excitation wavelength of 595 nm is suitable optical window for in vivo imaging 39.

The PCL-PEG-PPOR-PEG-PCL hydrogel erosion can be calculated from the total fluorescence decay of the hydrogel using the non-invasive CRI imaging system. The erosion determined by gravimetric analysis is generally faster than that by fluorescent tracking because the former considers the elution of materials within the network during swelling, in addition to the erosion of the polymer chains. Fluorescence tracking ensures the high- accuracy identification of polymer chains released from the hydrogel sequentially from the onset of hydrogel immersion without biotissue invasion and sample destruction. After injection, the erosion and degradation of the hydrogel can be non-invasively monitored from the decay of the total copolymer fluorescence signals. To track the in vivo erosion process of the hydrogel, whole animal imaging was performed

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on mice for three weeks (Fig. 3). In the dorsal subcutaneous injection group, the PCL-PEG-PPOR-PEG-PCL hydrogel forms a gel in situ from the liquid sol and the intense fluorescence of the hydrogel can be shown. With the permeation and distribution of the hydrogel, its fluorescence gradually shrinks. During the monitoring process of non-invasive fluorescence imaging, the protoporphyrin incorporated hydrogel maintains its form and location in vivo. The construct erosion of the hydrogel in vivo was calculated quantitatively from the total fluorescence signals in the efficiency region of the implanted hydrogel. As shown in the erosion curve (Fig .4), the erosion rate is rapid in the first week. The fluorescence strength exhibits a 12 % decrease on the first day. With continued erosion over the first week, the optical density still remains at approximately 44%. From then on, the erosion rate of the hydrogel begins to step down. After three weeks, it retains only a faint fluorescence signal of 10 % compared to the initial fluorescence.

To further study the organ distribution and the status of the hydrogel, the mice were sacriﬁced after three weeks. The dark red gel is viewed clearly under the skin in a picture taken 21 days after subcutaneous the injection (Fig. S6A). In the subcutaneous tissue of the experimental mice, the undegraded hydrogel still obviously exists and retains a strong fluorescence ex vivo (Fig. S6B). The fluorescence of the remaining hydrogel can be feasibly removed under imaging guidance. Key organs including the heart, liver, spleen, lung, kidney, and skin of the administration site were harvested for the analysis of the material biodistribution by fluorescence imaging ex vivo. The skin tissue of the administration site exhibits obvious fluorescence from the permeation of the hydrogel, while there are little fluorescence signals in the other organs (Fig. S6C). To investigate the morphology of the resected hydrogel after erosion, SEM was employed. The remained hydrogel is connected with the biotissue. The surface of the resected hydrogel shows inhomogeneous cracks and links, while the initial hydrogel presents a homogeneous mesh structure (Fig .S7). This difference in morphology is caused by the erosion from enzyme and other biomolecules. Histological analysis was performed to assess the in vivo biocompatibility and safety of the PCL-PEG-PPOR-PEG-PCL hydrogel on biotissue and key organs. According to HE staining shown in Fig. 5, the hydrogel produces no significant inflammatory reaction or histopathological changes in the skin of the administration site. The key organs also show no histopathological 14


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changes or abnormal damage, which confirms the good biocompatibility and safety of this implanted hydrogel in vivo.

3.5 Multispectral fluorescence imaging for drug release from hydrogel The release of the drug is the most crucial for the drug delivery system. Due to various limitation, in vitro evaluation may not represent the release effect in vivo sufficiently accurately. Non-invasive in vivo fluorescence imaging is a suitable techique for the monitoring the in vivo release of drug because the reduction of the fluorescence intensity at the implantation site largely reflects the release of the drug from the carrier51-53. It requires a much smaller number of animals than the other methods while still obtaining high- quality of data. Importantly, the interrelation of the drug and carrier can be observed in situ in a drug-loaded hydrogel system by multispectral fluorescence imaging, which can track two or more fluorescence probes simultaneously based on their differences in emission. Multispectral analysis is based on the fact that the spectrum is different for each specific fluorescent material. A multicolor composite image can be generated to distinguish difference labels. This is a unique function that other imaging techniques can not provide. Multispectral fluorescence imaging has been serviced for monitoring the biofate of biomaterials and tracking drug delivery 12,54,55. Multispectral fluorescence imaging can provide the location and status of the drug and carrier clearly.

To track the release behavior of the drug from the hydrogel and monitor its location, multispectral fluorescence imaging analysis was performed using rhodamine as a model drug. Comparison of in vitro and in vivo drug release from the hydrogel system was investigated. As shown in the release curve in vitro (Fig. 6), the release from the hydrogel was accelerated with approximately 40% on the first day at a fast release rate. Then the sustained release continued to the fifth day with an accelerated release of less than 60 %. Multispectral fluorescence imaging provided the drug release effect from the hydrogel in vivo. It can be assessed that the decay of the fluorescence intensity at the implantation site over time largely reflects the release of drug from the hydrogel using free rhodamine solution as a control. Due to the fluorescence difference betweem the

PCL-PEG-PPOR-PEG-PCL hydrogel and rhodamine, they can be clearly

distinguished in yellow and blue by the multispectral fluorescence techique, respectively (Fig. 7).

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From the in vivo imaging of drug delivery (Fig. 8), the free rhodamine is rapidly distributed after 24 hours without persistent retention at the administration site, such that only a faint blue fluorescence from the model drug is detected. The mixed fluorescence of yellow and blue from the drug-loaded hydrogel appears in its location. The drug undergoes a sustained release, shown in yellow, from the implanted hydrogel, shown in blue, for 5 days. To observe the drug release, the fluorescence imaging in vivo is compared using a rainbow scale bar (Fig. 9). The remaining fluorescence is still 9 % after 5 days owing to a significant slower release of drug, which is likely to reflect the network structure consisting of many pores and meshes in hydrogel. The release of the model drug rhodamine was assessed by fluorescence imaging in this study. It shows a conspicuous sustained release in comparison with free drug. Although the drug release in vivo shown a faster release than that in vitro at the same condition because of the difference of biological environment including enzyme and peotein, the trend of the drug release in vitro and in vivo proved the sustained process from the PCL-PEG-PPOR-PEG-PCL hydrogel. Comparison of in vitro and in vivo protein release from other hydrogel systems provided the similar phenomenon53. The drug release from the hydrogel can be clearly observed by multicolor imaging in vivo. Multispectral analysis separates the autofluorescence signals away from the specific labels, and provides the ability to locate drug and carrier. It would offer important information for the development of novel drug carriers.

4 Conclusions In summary, an amphipathic thermosensitive protoporphyrin-incorporated hydrogel based on PEG-PCL copolymer was successfully prepared by open ring polymerization. Protoporphyrin was incorporated into the backbone as a fluorescence tag. It adds fluorescence to the injectability of the hydrogel. The hydrogel erosion and drug delivery in vivo were monitored and tracked continuously by a multispectral fluorescence imaging system using nude mice as models. With the guidance of the fluorescence imaging, the status of the hydrogel can be observed in vivo, including its location and erosion. The thermosensitive hydrogel has stable fluorescence for real-time imaging and good biocompatibility. The PCL-PEG-PPOR-PEG-PCL hydrogel can be a viable biomedical implant for tissue repair or drug delivery. Multispectral fluorescence imaging provides a non-invasive tool for implanted biomaterials to be located, monitored and tracked in situ. 16


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Acknowledgement This work was supported by the National Natural Science Foundation of China (No.31200732) , Excellent Young Scientists Fund of NSFC (NO.NSFC31222023) and the Natural Science Foundation of Tianjin,China (No.14JCYBJC17400).

Associated Content: Supporting Information. Characterization of protoporphyrin Incorporated thermosensitive hydrogel.

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Figure legends Fig. 1 Synthetic route for PCL-PEG-PPOR-PEG-PCL copolymer Fig.2 UV-vis (A) and fluorescence PCL-PEG-PPOR-PEG-PCL copolymer

emission

(B)

and

excitation

spectra

(C)

of

Fig. 3 Fluorescence imaging of PCL-PEG-PPOR-PEG-PCL hydrogel in vivo at different times, for one representative of three in each group Fig. 4 Fluorescence analysis of hydrogel degradation Fig.5

Histological

sections

of

major

organs

after

subcutaneous

administration

of

PCL-PEG-PPOR-PEG-PCL hydrogel (A-F) and major organs of control group (G-L) (× 40) (A.F:heart; B.H:liver; C.I:spleen; D.J:lung; E.K:kidney; F.L:skin)

Fig. 6 Drug release from hydrogel in vitro Fig. 7 Fluorescence analysis of drug and hydrogel in vitro with rainbow color (A) and single color (B) Fig.8 Multispectral fluorescence imaging for drug release from hydrogel using mixed fluorescence of drug and hydrogel with blue and yellow, for one representative of three in each group Fig.9 Drug release from hydrogel with rainbow color (A) and quantitative analysis of fluorescence (B), for one representative of three in each group

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Fig. 1 Synthetic route for PCL-PEG-PPOR-PEG-PCL copolymer 125x75mm (300 x 300 DPI)


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Fig.2 UV-vis (A) and fluorescence emission (B) and excitation spectra (C) of PCL-PEG-PPOR-PEG-PCL copolymer 1378x346mm (96 x 96 DPI)



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Fig. 3 Fluorescence imaging of PCL-PEG-PPOR-PEG-PCL hydrogel in vivo at different times, for one representative of three in each group 312x190mm (96 x 96 DPI)


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Fig. 4 Fluorescence analysis of hydrogel degradation 55x38mm (300 x 300 DPI)



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Fig.5 Histological sections of major organs after subcutaneous administration of PCL-PEG-PPOR-PEG-PCL hydrogel (A-F) and major organs of control group (G-L) (× 40) (A.F:heart; B.H:liver; C.I:spleen; D.J:lung; E.K:kidney; F.L:skin) 343x192mm (96 x 96 DPI)


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Fig.6 Drug release from hydrogel in vitro 55x38mm (300 x 300 DPI)



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Fig. 7 Fluorescence analysis of drug and hydrogel in vitro with rainbow color (A) and single color (B) 370x137mm (96 x 96 DPI)


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Fig. 8 Multispectral fluorescence imaging for drug release from hydrogel using mixed fluorescence of drug and hydrogel with blue and yellow, for one representative of three in each group 423x296mm (96 x 96 DPI)



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Fig. 9 Drug release from hydrogel with rainbow color (A) and quantitative analysis of fluorescence (B), for one representative of three in each group 314x191mm (96 x 96 DPI)


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For Table of Contents Only 196x217mm (150 x 150 DPI)


Real-Time Fluorescence Tracking of Protoporphyrin Incorporated

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