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“one-for-all” Type, biodegradable Prussian Blue/Manganese Dioxide Hybrid Nanocrystal for Tri-modal imaging guided Photothermal Therapy and oxygen regulation of Breast Cancer JinRong Peng, Mingling Dong, Bei Ran, Wenting Li, Ying Hao, Qian Yang, Liwei Tan, Kun Shi, and ZhiYong Qian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 4, 2017

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ACS Applied Materials & Interfaces

“one-for-all” Type, Biodegradable Prussian Blue/Manganese Dioxide Hybrid Nanocrystal for Tri-Modal Imaging Guided Photothermal Therapy and Oxygen Regulation of Breast Cancer Jinrong Peng†, Mingling Dong†, Bei Ran†, Wenting Li‡, Ying Hao†, Qian Yang§, Liwei Tan †, Kun Shi†, Zhiyong Qian†,* †

State Key Laboratory and Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, No. 17, Section 3, Southern Renmin Road, Chengdu, Sichuan, P. R. China Sichuan, P. R. China. ‡

Department of pharmacy, West China Second University Hospital, No. 20, Section 3, Southern

Renmin Road, Chengdu, Sichuan, P. R. China §

School of Pharmacy, Chengdu Medical College, No. 783, Xindu Avenue, Xindu District,

Chengdu, Sichuan China.

*

To whom should be corresponded. Tel/Fax: +86-28-85501986, E-mail: [email protected] (QZY).

ACS Paragon Plus Environment

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Abstract

Multi-modal imaging guided diagnosis and therapy has been highlighted in the area of theranostic nanomaterials. In order to provide more suitable theranostic candidates, Prussian blue (PB) /manganese dioxide (MnO2) hybrid nanoparticles (PBMn) smaller than 50nm is prepared via a one-pot method. MnO2 which is reduced from KMnO4 not only control the particle size, the optical properties and the transverse relaxation rate (r2) of PB, but also enhance the catalysis efficacy of PB to H2O2 for oxygen generation. PBMn can be served as photoaccoustic imaging (PAI) and longitudinal relaxation (T1) mode magnetic resonance imaging (MRI) contrast agent (14 times and 1.8 times of saline treated group, respectively). Injection of PBMn can regulate the oxygen partial pressure of tumor tissue from 2.1±0.2 kPa to 9.3±0.4 kPa, and rearrange the ratio of oxygenated hemoglobin and deoxygenate hemoglobin inside tumor, which favor to enhance the diamagnetic T2 weighted imaging (T2WI) signal intensity (two times of saline treated group). Furthermore, PBMn mediated PTT can efficiently inhibit the growth of MCF-7 tumor in vitro and in vivo. PBMn can be served as PAI/T1/T2 tri-modal contrast agent, and for imaging guided PTT and oxygen regulation of the exografted breast cancer.

Keywords Multimodal imaging; photothermal therapy; hybrid nanoparticles; MRI; oxygen regulation


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Introduction The development of nanotechnology and nano-biomaterials provides infinite possibilities for cancer diagnosis and therapy.

1, 2

Not only efficient tumor site targeting diagnosis and therapy

can be achieved, but also the tumor microenvironment can be regulated by multifunctional nanoparticles. For their easy multifunctional modifications, nanoparticles have been designed and used to synchronously detect and treat cancer, which spurs the emergence and rapid progress of theranostic nanomedicines. 3 Although it is still in its early stage, theranostic nanomedicines have been received enormously attention for their diversity properties and combinations. The introduction of nanosystems as contrast agents indeed enhances the resolution and imaging quality of magnetic resonance imaging (MRI), positron emission computed tomography (PET), X-ray computed tomography (X-CT), fluorescent imaging (FI), etc.

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Among all these

techniques, MRI is an outstanding, non-invasive and relative harmless one.

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Numerous of

nanosystems which containing Fe, or Gd, or Mn, etc. have been constructed as MRI contrast agents. 11-14 Moreover, MRI can be used as a powerful tool to detect or study the diversity of the tumor microenvironment with the help of the contrast agents which can response to tumor microenvironment or specific biomarkers, such as pH, hypoxia, ions, or biomolecular biomarkers. 15-21 Mn based nano-contrast agents have been highlighted in this area. Its ionic state, Mn2+, can be used as T1 modal contrast agent. While combined with pH responsive nanocarriers, controlled release of Mn2+ and enhanced MRI of hypoxia tissue inside tumor can be achieved. 22 Moreover, its oxide form, manganese dioxide (MnO2), which is H2O2 sensitive， catalyze H2O2 to generate O2, and release of Mn2+, which act like as H2O2 responsive T1 contrast agent.

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The

generated O2 can reconstruct the oxygen supplying of the tumor, which not only helps to improve the hypoxia environment of the tumor and is critical to the tumor growth and metastasis


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inhibition,

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but also can turn the deoxygenated hemoglobin (Hb) to oxygenated hemoglobin

(HbO2). Deoxygenated hemoglobin is superparamagnetic while oxyhemoglobin is diamagnetic. The transformation of Hb to HbO2 can increase the transversal relaxation time (T2) and reduce transversal relaxation rate (r2), which make the tumor in T2WI image brighter.

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The

diamagnetic character of oxyhemoglobin or superparamagnetic of deoxygenated hemoglobin is also the theoretical foundation (BOLD (blood oxygenation level dependent) effect) of fMRI (functional MRI). Therefore, MnO2 can be served as T1-T2 dual-modal contrast agent for MRI as well as a promising candidate for oxygen production. 32, 33 Another critical important function of theranostic nanosystems is therapeutic functions, particular activatable therapeutic functions.

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Photomediated therapeutic manners are the

promising choice for activatable therapy. 36 From previously reports, photothermal therapy (PTT) not only can significantly inhibit the growth of cancer cells via photothermal ablation, but also can greatly enhance the chemotherapy efficacy as well as immunotherapy efficacy which combines with check-point blockage immunotherapy.

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Kinds of nanosystems have been

precisely designed and fabricated for the pre-clinical application in PTT of cancer.

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Combining with these nanomaterials, we can construct PTT based theranostic nanosystems in “all-in-one” or “one-for-all” forms.

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However, most of these nanomaterials are still facing the

obstacle of biodegradable and metabolic issues. And in the case of “one-for-all”, how to incorporate MnO2 into the PTT nanocarriers and efficiently form stable nanoparticles with suitable size is still an another main challenge to be overwhelmed. Prussian blue (PB), a FDA approved dyes to treat heavy metal toxicosis, can also be served as PTT nanocarriers for its strong absorption in near infrared (NIR) region, which is also can be used as PAI contrast agent. Different from some organic dyes, such as ICG, IR820 or IR780 etc.,


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PB is constructed by Fe(II) and Fe(III) complexes, and it is easy to be fabricated to nanostructures, and it is degradable.

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Moreover, incorporation of MnO2 to PB framework may

obtain T1 modal contrast agent. And some methods have been developed to cover MnO2 onto the PB framework, and obtained MnO2/PB nanostructure for MRI/PA dual modal imaging guiding therapy. 47, 48 However, most of these systems are constructed by two steps procedure. And to our knowledge, few reports about MnO2/PB nanostructure which were controlled synthesized with the particle size smaller than 50nm have been published. In generally, some reports have been claimed that the particle with the particle size ranging from 30nm to 200nm are believed to be optimal for passive targeting of most types of solid tumors by capitalizing on the enhanced permeability and retention (EPR) effect, passive targeting effects.

50-52

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and size reduction could be helpful to increase the

In order to obtain optimal passive targeting effect, it is still needs

to develop facile and simple method to fabricate the MnO2/PB nanostructure with the particle size smaller than 50 nm. Herein, we intend to use MnO2 which reduced from KMnO4 to confine the further growth of PB framework structure, and we have successfully fabricated the PB/MnO2 nanosystem with the particle size smaller than 50 nm by a one-pot controlled synthesis process, which can produce oxygen efficiently to oxygenate deoxygenated hemoglobin to enhance the diamagnetic T2 signal intensity in tumor and serve as T2 modal contrast agent as well as T1/PAI dual modal contrast agents (Scheme 1). Meanwhile, it also maintains its photothermal conversion for PTT. The controlled synthesis process, characterization and properties of the PBMn have been studied in details in vitro and in vivo.

RESULTS AND DISCUSSIONS


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Synthesis and Characterization of PBMn. Mn can form coordination with the CN groups which is highly containing in PB structure,

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therefore, we intend to use MnO2 to impede the

growth of PB framework structure. By this way to control the particle size of PBMn and introduce MnO2 into the PB framework (Figure 1A). By adding of KMnO4, KMnO4 was reduced to MnO2 in weak acidic conditions, which can attach to the growing PB nanoframework, and impede the further growth of PB (Figure 1B). As the increase of KMnO4 dose, the particle size of PBMn decreased from ~240 nm to ~50 nm which observed by TEM (Figure 1B). The size change of the PB also be studied using dynamic laser scattering (DLS) (decreased from ~400 nm to ~100 nm in Figure 1C). By optimizing the synthesis parameters, while the KMnO4 dose reached to 52 mg, PBMn with a particle size smaller than 50nm was obtained, which is named as PBMn-52 (Figure S1A). We have also calculated more than 200 numbers of PBMn-52 from the TEM images, the obtained particles size is 44.7±3.2nm. And the particle dispersion index of PBMn-52 dispersion obtained from DLS is 0.057±0.007. Therefore, PBMn-52 has a monodisperse distribution. SEM further proved its uniform distribution with a particle size smaller than 50nm, and the Fe, Mn elements distributed evenly on the SEM mapping images (Figure 1D and Figure S1H). The scanning transmission electron microscopy (STEM) further cleared the Mn and Fe distribution on a single particle. The Mn were coated around the Fe element, and the Mn/Fe ratios in the edge of the particle is much higher than the core, indicating the PB/MnO2 is a core-shell structure (Figure 1E and Figure S1G). The shell thickness and core size of PBMn-52 were 5±1.3nm and 39±4.2nm, respectively. The structure of MnO2 was further identified by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), as shown in Figure S1D, Figure 1F and S1E and S1F, respectively. The binding energy of Mn in PBMn is 654 eV and 641 eV, which concord with MnO2. And from XRD, the 2Θ at 38o, 42o, and 56o


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indicated the existence of MnO2. The results indicated that the PB/MnO2 hybrid nanosystem was successfully fabricated. PBMn-52 maintains its particle size after being dispersed in different mediums, including water, PBS (pH=7.4), saline, and serum, in 30min (Figure 1G). By DLS real-time measurement, the PBMn-52 maintained 96.7±8.6 nm hydrodynamic particles size with 0.065±0.012 polydispersity after 4 months of storage in water or normal saline compared to the just synthesized particles (101.3±6.2 nm). It indicates that the PBMn-52 is stable in water or saline. The Fe and Mn contents of PBMns were measured by ICP-AES, the results were listed in Table S1. No relationships were found between the doses of KMnO4 and the Fe and Mn contents in PBMn. Optical properties and photothermal conversion of PBMn-52. The introduction of MnO2 can affect the optical absorption of PB, as shown in Figure 2A. While the dose of KMnO4 was equal or lower than 26 mg, as the increase of KMnO4, the absorption of PBMn in NIR region decreased. While the KMnO4 was dosed at 52 mg, the PBMn-52 has similar absorption with PB. Further increase the dose of KMnO4, the absorption of PBMn in the NIR region can be further increased. The results indicated that as the increase of KMnO4 dose from 0 to 26 mg, the absorption of the obtained PBMn in NIR region decreased. The further increase of PBMn-26 from 26 to 104, the absorption of PBMn in NIR region were getting stronger. It suggests that the dose of KMnO4 not only can regulate the particle size of PBMn, but also affects the optical absorption. The underline mechanism of optical property variation with the dosage of KMnO4 is still unclear, it may be ascribed to the change of the PB framework structure after the introduction of MnO2. We also evaluated the effects of HCl and other Mn donors onto the absorption of PBMn in NIR region, and found out that in order to control the particle size and


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NIR absorption of PBMn, we needed to carefully adjust the doses of HCl and KMnO4 and their ratios, as shown in Figure S2A and S2B. The absorption of PBMn has critical connection with its photothermal conversion efficacy. After optimization, we choose PBMn-52 as the optimized condition for the further applications because its particle size is around 50nm and it has strong adsorption at NIR region. As the increase of PBMn concentration, the ∆T of the PBMn-52 dispersion can reach to 60oC when the concentration of PBMn-52 was 200 µg/mL. while the PBMn concentration was 50 µg/mL, the temperature of PBMn-52 aqueous dispersion can increase from 37oC to 62oC, indicated the high photothermal conversion efficacy of PBMn (Figure 2B). More important, the PBMn-52 remained stable even after the irradiation. After six circuits of on-off irradiation, the PBMn still has similar absorption in NIR region and still maintains its high photothermal conversion efficacy (Figure 2C and 2D). The results demonstrated that the PBMn-52 has efficient photothermal conversion, it is a promising candidate for PTT. Oxygen production from H2O2 by PBMn-52 catalyzing. Moreover, the introduction of MnO2 into the PB framework has also changed the catalytic efficacy of PB to H2O2. As shown in Figure 2E, the oxygen generated much faster in the PBMn-52 aqueous solution with the presence of H2O2 than in the PB solution. The oxygen production can reach to 15.78 mg/L while the PBMn-52 concentration was 50 µg/mL and H2O2 concentration was 100 µM in 45 s, much higher than PB did (7.89 mg/L). It has been proved that the deficit of oxygen may result in promoting the growth of tumor and tumor metastasis. 25 The production of oxygen may help to rebalance the oxygen level in the tumor. And the maintaining of oxygen in a normal level inside the tumor may efficiently inhibit the cancer growth and metastasis. 28 Therefore, for their high efficacy in H2O2 reduction, PBMn can be a satisfactory oxygen supplier, particularly in the area


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with high H2O2 expression. It has been reported that tumor tissues and tumor cells are all expressed much higher level of H2O2 than normal tissues and normal cells.

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Therefore,

PBMn can be used to regulate the oxygen environment inside tumor. The bubble existed in the inset photographs in Figure 2F further supported the ability of PBMn in catalyzing H2O2 to O2. And in the presence of H2O2, the adsorption of PBMn-52 had little change in NIR region, which indicated the photothermal conversion was not affected by the presence of H2O2 (Figure 2F). Relaxation rate (longitudinal relaxation rate (r1) and r2) modulation of PB by MnO2. MnO2 can be served as T1-T2 dual modal MRI contrast agent. PB, however, contains relative high content of Fe, particular the trivalent Fe, which may have short T2 and high r2 value (superparamagnetic). And the incorporation of MnO2 into PB framework may affect the relaxation rate of PB. Therefore, we have studied the effect of MnO2 on the modulation of relaxation rate of PB. By adjusting the dose of KMnO4, we revealed that the introduction of MnO2 significantly reduced the r2 value of PB from 89 mM-1 s-1 to 15 mM-1 s-1, such a low r2 value may have neglectable effect on the signal intensity in T2 MRI, meanwhile, the r1 value of PB increase from 0.5 mM-1 s-1 to 4.9 mM-1 s-1, see Figure 2G-J and Figure S3. It indicated that control the dose of KMnO4 could modulate the r2 and r1 value of PBMn. Furthermore, it has been noticed that the expression level of H2O2 in tumor tissues or tumor cells are much higher than the normal tissues or normal cells, 53, 54 then we subsequently evaluated the T1 and T2 signal variation of PBMn with the presence of H2O2. Under the H2O2 condition, we observed a dramatic decrease of r2 value from 89 mM-1s-1 to 11.4 mM-1s-1 for PB, and from 15.2mM-1s-1 to 28.3 mM-1s-1for PBMn-52, respectively, indicating the presence of H2O2 could increase the r2 value of PBMn-52, see Figure 2K-N. Further increase of the H2O2 concentration had little effect onto the r2 values of PBMn-52 (19.4 mM-1s-1, 300mM of H2O2), but increased the r1 value (32.4


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mM-1s-1, 300 mM of H2O2), as shown in Figure S4A-B. But both the r1 and r2 value hardly changed at the H2O2 ranged from 0.1mM to 10mM, see Figure S4C-D. And as the incubation time increase, the T2 MRI signal intensity increased, see Figure S4E. Although the r2 of PB dramatically reduced to 11.4 mM-1s-1 in the presence of H2O2, its larger particle size can weaken its accumulation in tumor via EPR effect. The r2 of PBMn-52 has a little increase in the presence of H2O2, but its inherent r2 is small and is still very small in the presence of H2O2, much smaller than the reported commercial T2 contrast agents.

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Combining with its excellent properties as

T1 contrast agent and it high catalytic efficacy in oxygen generation from H2O2, PBMn-52 is still a promising candidate for T1 and enhanced T2 diamagnetic MRI. Cell viability, cellular uptake and photothermal therapeutic efficacy in vitro. Before its application in vivo, we first evaluated the cell viability of different cell lines after incubating with PBMn and the anticancer performance of PBMn-52 mediated PTT in vitro. We tested the cell viability of different cell lines after incubating with blank PBMn-52 in vitro. The cell lines included 3T3, 4T1, and MCF-7. As shown in Figure S6A, while the concentration of PBMn-52 reached to 1.0 mg/mL, the cell viability of all the cell lines is still higher than 90%. And by hemolysis test, no hemolysis or coagulation were observed, as shown in Figure S5. Due to the structure of PBMn-52, it is hard to label it with fluorescent dye to evaluate the cellular uptake. So we used an indirectly way. The Mn can quench the fluorescence of calcein-green; therefore, we used calcein-green to study the cellular uptake behavior of PBMn-52 in vitro. As shown in Figure S6B, after the incubation with PBMn-52 for 4h, the cancer cells were stained with calcein-green, compared to the control group, the fluorescence of the cancer cells incubated with PBMn-52 is significantly quenched. It indicated the PBMn-52 were engulfed into the cancer cells. We further evaluated the PTT of PBMn-52 in vitro. 4T1 cells and MCF-7 cell lines were


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chosen as the model cancer cell lines. Figure S6A is the results of the viability of the cells after being treated with PBMn-52 under the irradiation of 808nm laser in vitro. Apparently, while the concentration of PBMn-52 is higher than 50 µg/mL, the growth of the cancer cells was significantly inhibited, which is further proved by the calcein-green/DAPI dual staining of the cancer cells after laser irradiation, as shown in Figure S6C. It indicates that PBMn-52 is a promising candidate for cancer cells PTT. PAI and T1 modal MRI in vivo. Further, we investigated the efficacy of PBMn-52 as contrast agents for PAI and T1 modal MRI in vivo. After intravenously injection of 10 mg/kg b.w. of PBMn-52, in two hours later, comparing with PB group and saline groups, PA signal of the tumor tissue was dramatically increased, which indicated the PBMn-52 has enriched in the tumor. And as the time prolonged, the signal increased. After 24 hrs, the PA signal of the tumor treated by PBMn-52 is 12 fold higher than PB, and 20 fold higher than NS group, as shown in Figure 3A-B and Figure S7A-B. The results indicated that PBMn-52 can be served as PA contrast agent. Then we evaluated the efficacy of PBMn-52 as T1 modal MRI contrast agent. After the intravenously injection of 10 mg/kg b.w. of PBMn-52, in two hours later, comparing with the NS treated group, the T1WI signal intensity dramatically increase to a highest value. And as the time prolonged, the T1WI signal intensity decreased, as shown in Figure 3C-D. The decrease of T1WI intensity may be ascribed to the metabolism of Mn2+ which released from MnO2 or the PBMn-52 particles could have been cleared away from the tumor as time prolonged. From the in vitro MRI measurement, we can conclude that the PBMn-52 can be used as T1 modal contrast agent. Oxygenated Hb mediated T2 MRI in vivo. MnO2 can catalyze H2O2 to O2, and oxygenate deoxygenated Hb to HbO2. And HbO2 is diamagnetic. However, PB is paramagnetic with r2


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value of 89 mM-1s-1. The introduction of MnO2 to the PB framework decreased the r2 value of PBMn-52 (15.4 mM-1s-1). Therefore, the influence of PBMn-52 onto the diamagnetic imaging of HbO2 can be diminished. And it has been reported that the tumor tissues or tumor cells expresses more H2O2 than the normal tissues or normal cell. The high efficient of PBMn-52 in catalyzing H2O2 to generate oxygen may help to further oxygenate the deoxygenated Hb to HbO2, which makes the PBMn-52 serve as T2 diamagnetic contrast agent. After intravenous injection of 10mg/kg b. w. of PBMn-52 in vivo, compared with PB treated group and NS treated group, the T2WI signal intensity dramatically increase, see Figure 4A-B and Figure S7b. It indicates the PBMn-52 can be also served as a diamagnetic T2 modal contrast agent. We further evaluated the HbO2 value of the tumor after the treatment of PBMn-52 by PAT. After the administration PBMn-52, the HbO2 intensity in PAT images is 3 fold of PB treated group and 14 fold of NS group, see Figure 4C-D and Figure S7b. The oxygen partial pressure of the tumors recorded by polarograph microelectrodes revealed that the oxygen partial pressure of the tumor which treated by intravenous injection of PBMn-52 increased from 2.1±0.2 kPa to 9.3±0.4 kPa, while the saline treated group remained unchanged (Figure 4E) And by immunofluorescence staining with DAPI and hypoxia marker of the tumor sections, we further revealed that PBMn-52 can regulate the hypoxia condition of the tumor (Figure 4F). It reveals while the PBMn-52 enriched into the tumor tissue, it may catalyze the high expression of H2O2 to O2, and then the O2 oxygenated the deoxygenated hemoglobin (which is highly containing in tumor site for its hypoxia microenvironment) to oxyhemoglobin. As we known, the deoxygenated hemoglobin is superparamagnetic, and oxyhemoglobin is diamagnetic. Deoxygenated hemoglobin has lower T2WI signal intensity, while oxyhemoglobin has higher T2WI signal intensity. Therefore, the enrichment of PBMn-52 in tumor site has directly increased the T2WI signal intensity for oxygen


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generation. It demonstrated that the PBMn-52 has highly response to H2O2 and can be used as oxygen catalyzer and a diamagnetic T2 MRI contrast agent for H2O2 identification. PBMn-52 mediated Photothermal therapy of breast cancer in vivo. Finally, before we treated the breast cancer by PTT in vivo, we first investigated the photothermal conversion of PBMn-52 in vivo. MCF-7 breast cancer model were established on balb/c nude mice. As shown in Figure 5A, in PBMn treated group, the temperature of the tumor area which was irradiated by 808nm laser raised up to 72oC in 5min, while the control groups hardly changed. And the photothermal conversion is PBMn-52 dose-dependent, as shown in Figure 5B. While the dose of PBMn-52 is 10 mg/Kg body weight, the temperature raised up to 54oC degree. Moreover, we also semi-quantitatively evaluated the depth of the heat transfer in the tumor tissue under the laser irradiation. As shown in Figure 5C, less than 5min of indication, the depth of the tumor tissues which the temperature is higher than 45oC only reached to 4 mm. It demonstrated that the PBMn mediated PTT can only penetrate 4mm of tumor tissue. It may ineffective while the tumor thickness is thicker than 4 mm. In order to prove that, we have further investigated the effect of primary tumor volume onto the efficacy of PBMn mediated PTT. While we treated the the tumor with the primary volume of ~125 mm3, under PTT, all of the tumors treated by PBMn-mediated PTT were totally eliminated , as shown in Figure 5D-F and Figure S8A. And the mice were tumorfree from day 17 to day 90 (the observation period lasted for 90 days, at day 90, all the mice were killed). If the primary tumor volume reaches to 450 mm3 while we begin the PTT, no tumor can be eliminated in PBMn-52 mediated PTT treated group. PTT just slowed down the growth of the tumor, as shown Figure S8B-D. It indicated the primary tumor has critical effect onto the PBMn mediated PTT. This is also concord with the results of the depth of the heat transfer. Furthermore, by investigating the biodistribution of PBMn in vivo, by measuring the Mn


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content in the tissues via inductively coupled plasma mass spectrometry (ICP-MS), we revealed that the Mn were highly enriched in tumor tissue in 1h, and it mainly distributed in liver and kidney, as shown in Figure 5G. As the time prolonged, the Mn in most of the organ (heart, spleen, lune, kidney, and tumor) were all reduced to normal level except liver. And by evaluating the degradation behavior of PBMn-52 in serum, we confirmed that PBMn can take place degradation in physiological environment (Figure S9). It demonstrated that the PBMn-52 is metabolic. Conclusion In conclusion, we used KMnO4 to one-pot controlled synthesized of Prussian blue/manganese dioxide hybrid nanoparticles (PBMn) for tri-modal imaging guided PTT and oxygen regulation. The introduce of MnO2 not only decreased the particle size of PB from 240 nm to 50 nm and the r2 value of PB and make the PBMn-52 served as PAI and T1 modal MRI contrast agent, but also enhanced the catalysis efficacy of PB to H2O2 for oxygen generation. The decrease of r2 has diminished the inherent artifact T2WI signal of PB for diamagnetic T2WI MRI. And the oxygen generation could rearrange the ratio of oxygenated hemoglobin and deoxygenate hemoglobin inside tumor, which also favored to enhance the diamagnetic T2WI signal intensity. It makes the PBMn-52 to be a diamagnetic T2 modal MRI contrast agent. By PAT and MRI evaluation, all the results have supported the PBMn-52 can serve as tri-modal imaging contrast agent. And it also is an oxygen regulator inside tumor. Further, the photothermal conversion properties of PBMn-52 have been remained. And by in vivo tumor growth inhibition trials, we revealed that the PBMn is a promising candidate for tri-modal imaging guided photothermal therapy and oxygen regulation.


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Experimental and methods Materials Poly(vinyl pyrrolidone) (Mw=40000), agar, intralipid, methyl thiazolyl tetrazolium (MTT), Calcein, and DAPI were all purchased from Sigma-Aldrich (Saint Louis, USA). Methoxy polyethylene glycol amine was purchased from Jenkem Technology CO., LTD (Beijing, China). KMnO4, potassium hexacyanoferrate(III) (K3[Fe(CN)6]), hydrochloric acid (HCl), H2O2, and MnCl2 were purchased from Tianjin Bodi Chemical Co. Ltd. (Tianjin, China). All the chemical reagents were used without further purification. Cell lines including 4T1 cells, MCF-7 cells, 3T3, HUVEC were all purchased from American Type Culture Collection (ATCC, Rockville, MD), which were grown in DMEM supplement with 10% of FBS, 100U/mL of penicillin and 100 µg/mL of streptomycin, respectively. The cell cultures were maintained in a 37oC incubator with humidified 5% of CO2 atmosphere. Female BALB/C-nu mice (4-6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and kept under SPF condition with free access to standard food and water. All animal procedures were performed following the protocols approved by Institutional Animal Care and Treatment Committee of Sichuan University (Chengdu, P. R. China). Synthesis and characterization of PBMn Synthesis of PBMn


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PB and PBMn was synthesized via hydrothermal reaction which is according to the previous reports with some modification. [S1, S2] Briefly, a typical synthesis procedure of PBMn-52 as following: 3 g of PVP was first dissolved in deionized water which containing 104 µL of concentrated HCl (36%, w/v) in room temperature. Then 132 mg of K3[Fe(CN)6] and 52 mg of KMnO4 were added sequentially into the PVP solution. After the KMnO4 was totally dissolved, the obtained brow-black solution was transferred to an oil bath which was preheated to 86oC immediately. The reaction was maintained for 24h. The obtained reaction mixture was centrifuged to collect the PBMn nanoparticles (16000 rpm, 30 min). The PBMn nanoparticles were washed by water for five times to remove the PVP. The purified PBMn nanoparticles were re-dispersed in water and stored at room temperature. During the optimization, different doses of concentrated HCl and KMnO4 were used to obtain a PBMn nanoparticles with satisfactory particle size and NIR absorption, while the doses of PVP and K3[Fe(CN)6] were fixed. Characterizations of PBMn Particle size distribution. The particle sizes of the obtained PBMn was measured by Dynamic laser scattering measurement (DLS, Nano-ZS 90, Malvern Instruments, Malvern, UK). The PBMn solution was diluted to 60ug/mL for the DLS measurement. Micromorphology. The morphological and structural information of PBMn were measured by a field-emission high-resolution transmission electron microscope (HRTEM, Tecnai G2 F20 STWIN). The diluted PBMn was drop onto the copper grid covered with nitrocellulose, and dried at room temperature on a filter paper. For the STEM measurement, the PBMn was drop and dried onto a carbon grid.


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Surface morphology. The surface morphology of the PB and PBMn was observed by scanning electronic microscopic (SEM, JSM-7500F, JEOL, Japan). The PBMn was lyophilized before the SEM observation. Energy Dispersive X-Ray Spectroscopy (EDX) and Mapping were used to semi-quantitatively measure the Fe and Mn contents and their distribution on the nanoparticles. Crystallization. The crystallization of PB or PBMn was evaluated by X-ray diffraction (XRD, EMPYREAN, PANalytical B.V., Netherlands). Molecular Binding Energy. The binding energy of the Fe and Mn in PB or PBMn structure was measured by X-ray photoelectron spectroscopy (XPS, AXIS Ultra DLD, Kratos, UK). Optical Absorption. The optical absorption of PBMn was measured by UV-visible spectrometer (PE, USA). Briefly, all the samples were diluted to 50 µg/mL before the measurement by UV-visible spectrometer. The wave length range was from 300 nm to 1100 nm, scanning speed was settled at 600 nm per minute, the absorption values were recorded every 5 nm. Composition. The content of the Fe and Mn element in PBMn was measured by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-MS, SPECTRO ARCOS, Spectro, Germany). Photothermal conversion in vitro The photothermal conversion of PBMn in aqueous solution was detected by thermometer with a probe. The PBMn aqueous solution was added into a EP tube with the maximum volume of 1.5mL. Then the probe of the thermometer was immersed into the PBMn solution. And the


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temperature of the initiated temperature was recoded. Under the 808 nm laser irradiation, the temperature of the PBMn solution detected by the thermometer were recorded at a predetermined interval. To evaluate the stability of the photothermal conversion of PBMn, and ON/OFF cycle irradiation experiment was processed. The PBMn solution was irradiated by 808 nm laser for 5min, then turn off the laser, and recorded the temperature variation. While the temperature of the PBMn solution dropped to the initiate temperature, the laser was turned on again. This process was repeated for six cycle, and the temperature variation was recorded. The variation of the UV-vis spectrums of the PBMn-52 before and after the irradiation was measured by UV-vis spectrometer. MRI signal enhancement of PBMn in vitro The MRI signals of the PBMn in aqueous solution were measured by Animal MRI instrument (BioSpec70/20USR, Bruke). Magnetic field strength is 7T. RARE sequence for T1, turboRARE sequence for T2. The PBMn samples were diluted to different concentration. The effect of H2O2 onto the MRI signal of PBMn was also investigated as the same procedure. Oxygen production by PBMn catalysis The oxygen generated by PBMn was measured by a Dissolved Oxygen Meter (JPB-608, Shanghai Instrument Electric Science Instrument Limited by Share Ltd.). Briefly, in a sealed double bottle neck flask (which one neck was containing a probe of dissolved oxygen meter immersing in the near bottom of the flask, and sealed with rubber plug), 100 mL of deoxygenated water was injected. Then a certain amount of H2O2 (30%, w/v) was injected into the flask to obtain a H2O2 solution with a concentration of 0.1mM. The PBMn dispersion was


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injected into the flask after the record of the detector became stable. The flask was maintained gentle shaking during the experiment. The values of the dissolved oxygen detected by the probe were recorded at predetermined times. Cell viability and hemolysis test Two cancer cell lines, 4T1 cells and MCF-7 cells were used to evaluate the photothermal ablation of PBMn in vitro. The cancer cells were seeded at 5 × 103 cells per well in a 96-well plate, pre-incubated for 24 h, then incubated with PBMn-52 for 4 h at the concentrations ranging from 0 to 500 µg/mL. The hemolytic study was performed in vitro according to our previous reports, but with some modification [40]. Briefly, the prepared PBMn-52 in saline were diluted to 2.5 mL using saline and then added to 2.5 mL rabbit erythrocyte suspension (2%) in saline at 37 °C. Saline and distilled water were used as the negative and positive controls, respectively. After 3 h, the erythrocyte suspension was centrifuged and the color of the supernatant was compared with the controls. Also, the absorption of the supernatant at 570 nm was measured by UV-Vis spectrophotometer, the hemolysis ratio was calculated by comparing with the positive control. Photothermal ablation of cancer cells in vitro Two cancer cell lines, 4T1 cells and MCF-7 cells were used to evaluate the photothermal ablation of PBMn in vitro. The cancer cells were seeded at 5 × 103 cells per well in a 96-well plate, pre-incubated for 24 h, then incubated with PBMn-52 for 4 h at the concentrations ranging from 0 to 500 µg/mL. Four hours later, the culture medium of each sample was replaced with fresh RPMI 1640 culture medium with penicillin-streptomycin after PBS buffer raising for one


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time. Then the 808 nm laser was introduced. The power of the laser was 2.5 W/cm2, irradiation time was 5 min. The Calcein/DAPI co-staining was performed immediately after the irradiation. The fluorescent images of the cancer cells were obtained by a fluorescent microscopy (Olympus, Japan). To investigate the effect of irradiation to the survival of the cancer cells, the cancer cells were further cultured for 24h after the laser irradiation. MTT assay were performed Photothermal conversion in vivo MCF-7 tumor bearing balb/c-nu mice were used to evaluate the photothermal performance of PBMn-52 in vivo. After the intravenous administration of NS, or PBMn-52, the mice were anesthetized and then irradiated by an 808 nm laser with a power of 2.5 W/cm2 for 5 min. The temperature of the tumor site was recorded by an infrared imaging device (Fluke, T32, USA). Phototheraml therapy in vivo The MCF-7 tumor model was established in Balb/c-nu mice. After the mean volume of the tumors reached approximately 125 mm3, the tumor-bearing mice were randomly divided into 4 groups (n = 5), then intravenously administered with NS, and blank PBMn-52 for the other three groups. Laser irradiation was introduced in two of three groups, laser power was settled at 1.8 W/cm2 and 2.5 W/cm2, respectively, irradiation time was 5 min. Tumor volumes and body weights were measured every other day. In order to evaluate the effect of the primary tumor volume onto the photothermal efficacy of PBMn-52, another animal trial was performed, which has similar procedure with the modification of the primary tumor volume at the beginning of the therapy changed from 125 mm3 to 450mm3.


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MRI in vivo Before the MRI, the balb/c-nu mice were anesthetized with chloral hydrate (10%, w/v). The MRI images of the mice before the PBMn-52 administration were taken. Then the mice were divided into two groups, and injected with normal saline, and PBMn-52 respectively. At predetermined time interval, the mice were anesthetized again for MRI observation. Magnetic field strength is 7T. Flash sequence for T1WI and turboRARE for T2WI were used. PAI in vivo and in vitro MCF-7 cancer model was established on balb/c-nu mice 10 days before PAI. PAI was measured by a Multi-Spectral Optoacoustic Tomography (MSOT, MSOT inVision128, iThera Medical GmbH, Germany) system. Laser frequency was 10Hz with six wavelengths which are 700, 715, 730, 760, 800, 850 nm. The step length was 0.3mm, speed of sound was -36. The size and resolution of the images were 25mm and 75um, respectively. The temperature of the sink was 34oC. Model-linear and linear regression were chosen as the reconstruction method and MSP method, respectively. The spectrums of Hb, HbO2 and the samples were used as MSP spectrums. The MSP background wavelength was 800nm. An artifact implant (consist of agar and intralipid) was used to measure the PAI intensity of the sample in vitro. Measurement of Oxygen Partial pressure of the tumor in vivo. The oxygen partial pressure of the tumors recorded by polarograph microelectrodes (pO2 ESeries Sensor, Oxford Optronix Ltd., UK). First, the mice were anesthetized. Before the PBMn52 injection, the microelectrodes were inserted into the tumor. The depth of the microelectrode


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inserted into the tumor was about 3 mm (the tumor size was ~10*10mm). The oxygen partial pressure values were recorded every single hour for four hours before the administration of PBMn-52. Then the mice were divided into two groups and treated by saline and PBMn-52 intravenously, respectively. The oxygen partial pressure was recorded timely. Statistic Analysis Statistical analysis was performed using SPSS 15.0 software (IBM Corporation, Armonk, NY, USA). The results were indicated as mean ± SD. ANOVA was employed for multiple group comparisons, and results of p < 0.05 were considered statistically significant.


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Scheme 1 Scheme of the multifunctionality of PBMn nanaoparticles. The introduction of KMnO4 can decrease the particle size of PB from 240 nm to ~50 nm, at the same time, enhance the catalysis efficacy of H2O2 to oxygen and decrease the r2 value of PB. The oxygen generated by PBMn catalysis can oxygenate the deoxygenated hemoglobin (which is highly containing inside the solid tumor, and is also superparamagnetic) to oxyhemoglobin (which is diamagnetic). It can increase the T2WI signal intensity while the deoxygenated hemoglobin is oxygenated to oxyhemoglobin. Besides, the PBMn has strong absorption in NIR region. It also has strong photothermal conversion efficacy.


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A

Figure 1 A) scheme of the confined effect of MnO2 on the growth of PB framework. B) TEM images of PBMn synthesized under different KMnO4 doses. Scale bar: A, upper, 200nm, bottom, 50nm. C) particle size distribution of PBMn synthesized under different KMnO4 doses. D) SEM images of PB and PBMn-52. Scale bar: 100nm. E) STEM images of PBMn and the ratio of Mn/Fe at point 1, 2, 3, respectilvey. F) the XPS spectrums of PB and PBMn-52. G) particle size distribution of PBMn-52 in different mediums.


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Figure 2 A) the UV-vis spectrums of PBMn synthesized under different KMnO4 doses, inset: the optical appearance synthesized under different KMnO4 doses. B) the photothermal conversion of PBMn-52 under the irradiation of 808 nm laser. C) photothermal conversion of PBMn-52 in aqueous solution (60 µg/mL) over six ON/OFF cycles of 808 nm laser, laser power: 2.5W/cm2. D) UV-vis spectrums of PBMn-52 in aqueous solution before and after six rounds of ON/OFF irradiation. E) the UV-vis spectrums of PBMn-52 in aqueous solution with the presence of H2O2 (100 µM). F) the oxygen catalyzed by PB and PBMn-52 with the presence of H2O2 in aqueous solution. G) and I) are the are the T1WI and T2WI MRI images of PB and PBMn-52 in aqueous solution in vitro, respectively, without the presence of H2O2, H) and J) are the linear association of Mn and Fe contents and 1/T1, 1/T2, respectively, without the presence of H2O2. K) and M) are the T1WI and T2WI MRI images of PB and PBMn-52 in aqueous solution in vitro, respectively, in the presence of H2O2 (60mM). L) and N) are the linear association of Mn and Fe contens and 1/T1, 1/T2, respectively, in the presence of H2O2 (60mM).


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Figure 3 PBMn mediated imaging guiding in vivo. A) PAT images of MCF-7 bearing mice in vivo before and after the intravenous administration of PBMn-52. B) the MSOT intensity variation of the tumour tissue vs time in vivo. C) T1WI MRI images of MCF-7 bearing mice in vivo before and after the intravenous administration of PBMn-52. D) The T1 signal intensity of the tumour tissue vs time in vivo.


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Figure 4 A) PAT images of HbO2 in MCF-7 bearing mice before and after intravenous administration of PBMn-52 in vivo. B) HbO2 PAT intensity variation of tumor tissue vs time in vivo. C) the T2WI MRI images of the tumor-bearing mice before and after the administration of PBMn-52. D) the T2 signal intensity of the tumour tissue vs time in vivo. E) dynamic oxygen partial pressure of the tumor treated by saline and PBMn-52, respectively, healthy muscle is also recorded as control. The arrow represents the beginning of the treatment. F) representive immunofluorescence images of tumor sections stained with DAPI and hypoxia marker (Hypoxyprobe™-1 Kit)


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Figure 5 A) the photothermal conversion of PBMn-52 in vivo. B) the relationships of the PBMn-52 dose and the photothermal conversion efficiency in vivo. C) the thickness of the tumor tissue which the temperature higher than 45oC under the irradiation of 808 nm laser. D) the tumor volume variation under the PBMn mediated PTT of MCF-7 breast cancer in vivo. E) the photographs of the tumor bearing mice took 2 weeks after the treatment. F) the body weight variation of MCF-7 bearing mice with different treatments. G) the biodistribution of Mn from PBMn in vivo.


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ASSOCIATED CONTENT Supporting Information. Some structural characterization, optical properties, imaging information and tumor growth inhibition in vitro and in vivo have been listed in Supporting information. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Author Contributions

Funding Sources This work was financially supported by the National Natural Science Fund for Distinguished Young Scholar (NSFC31525009), National Natural Science Funds (NSFC31222023 and NSFC31500809), Sichuan Innovative Research Team Program for Young Scientists (2016TD0004). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Dr. Shanling Wang from Analytical & Testing center, Sichuan University, P. R. China for the TEM observation and analysis of the data.


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Table of Contents.


39

Manganese Dioxide

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