肿瘤裸眼可视影像技术

1. Introduction For many forms of cancer, surgery is an essential treatment modality. The precise delineation of tumor margins and sub- sequent complete surgical resection, without damaging crucial Hydrogel Nanoparticles with Coomassie Blue for Brain Tum to the Surgeon Guochao Nie , Hoe Jin Hah , Gwangseong K Tanvi S. Ratani , Panagiotis Fotiadis , Ambe Thomas Chen , Daniel A. Orringer , Oren Sagh structures near the tumor bed, is one of the great challenges in brain tumor surgery. [ 1 ] The extent of tumor resection relies on the precision of tumor delineation and affects the length and quality of survival for the cancer patient. [ 2 ] In current clini- cal settings, resection of the tumor is guided by preoperative imaging studies, along with the operating surgeon’s ability to distinguish the lesion grossly by its appearance and con- sistency, with respect to normal tissue, which, by itself, is not reliable enough to achieve complete resection. In particular, brain tumor tissue, which is easily detected radiographically, Dr. G. Nie , [+] Dr. H. J. Hah , [+] Dr. G. Kim , [+] Dr. Y.-E. Koo Lee , M. Qin , T. S. Ratani , P. Fotiadis , A. Miller , A. Kochi , D. Gao , Prof. R. Kopelman Department of Chemistry University of Michigan Delineation of tumor margins is a critica cancer surgery. A tumor-targeting deep- agent is described, which, for the fi rst time, targeting, respectively. may be virtually indistinguishable from normal brain tissue in its visual appearance. Various attempts for improvement of tumor delinea- tion during brain tumor surgery have been made, based on two types of approaches: 1) implementing medical imaging instrumentation, and 2) applying fl uorescent or visual con- trasting reagents. The fi rst approach includes the imple- mentation of image-guided stereotactic navigation [ 3 ] and intraoperative magnetic resonance imaging (MRI). [ 4 ] These high-tech instrumentation-aided techniques have been used in the clinic and can greatly improve the accuracy of surgery. However, their use still has various restrictions, including possible reduced accuracy due to the practicing surgeon’s 930 N. University Ave., Ann Arbor, MI 48109, USA E-mail: kopelman@umich.edu T. Chen , Dr. D. A. Orringer , Dr. O. Sagher Department of Neurosurgery University of Michigan 1500 E. Medical Center Drive, Ann Arbor, MI 48109-5338, USA Dr. M. A. Philbert School of Public Health University of Michigan 1415 Washington Heights, Ann Arbor, MI 48109-2029, USA Dr. G. Nie Department of Chemistry and Biology Yulin Normal University Yulin, Guangxi 537000, China staining. This technology thus enables co with no need for extra equipment or speci agent consists of polyacrylamide nanopart molecules (for nonleachable blue color with polyethylene glycol and F3 peptides f © 2012 Wiley-VCH Verlag Gmb DOI: 10.1002/smll.201101607 [ + ] These authors contributed equally to this work. small 2012, DOI: 10.1002/smll.201101607 Covalently Linked or Delineation Visible im , Yong-Eun Koo Lee , Ming Qin , r Miller , Akiko Kochi , Di Gao , er , Martin A. Philbert , and Raoul Kopelman * l and challenging objective during brain blue nanoparticle-based visible contrast offers in vivo tumor-specifi c visible color lor-guided tumor resection in real time, al lighting conditions. The visual contrast icles covalently linked to Coomassie Blue contrast), which are surface-conjugated or effi cient in vivo circulation and tumor Cancer Therapy 1H & Co. KGaA, Weinheim wileyonlinelibrary.com divided attention between patient and monitor, longer setup time, high cost, limited availability, as well as poor instrument compatibility due to the necessity of having strong magnetic G. Nie et al. 2 full papers the NP and the F3 peptide, for improved stability in blood plasma and for preventing nonspecifi c binding. The new NPs, with covalently loaded CB, showed a highly selective blue staining of 9L glioma cells with negligible nonspecifi c staining in vitro, as well as selective tumor staining in vivo, in Figure 1 . Signifi cant nonspecifi c cell staining by dye leaching from CB-post-loaded PAA NPs. Brain tumor cells (9L gliosarcoma) incubated for 1 h with A) F3-targeted CB-post-loaded PAA NPs and B) nontargeted CB-post-loaded PAA NPs exhibit a strong positive blue staining effect regardless of cell binding of NPs by F3 targeting. Scheme 1 . Synthesis of CB-APMA: i ) POCl 3 , dimethylformamide (DMF), CH 2 Cl 2 , 40 ° C, 7.5 h/ ≈ 20 ° C, overnight; ii ) APMA, dimethyl sulfoxide (DMSO), triethylamine, CH 2 Cl 2 , 0 ° C, 30 min/ ≈ 20 ° C, 1.5 h. H3CH O NH3C N H3C N H O CH3 S NH O O H3C CH3 S O O N H N H O CB CB-intermediate (CB-I) CB-linked APMA (CB-APMA) ii 7 6 5 4 3 2 1 N H OOH- fi elds within the operating space (intraoperative MRI). The second approach has been attempted by using fl uorophores or visible dyes to stain tumor tissue. Fluorescein, [ 5 ] 5-aminole- vulinic acid (5-ALA), [ 6 ] indocyanine green, [ 7 ] bromophenol blue, [ 8 ] and Coomassie Blue [ 9 ] have been suggested. In prin- ciple, dyes can preferentially stain malignant gliomas as they diffuse more readily across the areas of breakdown (fenes- trations) of the blood–brain barrier. Although this approach allows surgeons to fully focus on the patient due to the visual contrasting effect coming directly from the tumor tissues, it is still subject to several limitations. For instance, fl uorescence dye-based delineation requires special lighting and the ele- vated risk in performing operations in near darkness, as well as interruptions of surgery. In addition, a general drawback of dye-based delineation has been a lack of target specifi city, systemic cytotoxicity, a requirement of intolerable high doses to achieve satisfactory visual contrast, and short-lasting reten- tion at the desired site. [ 10 ] The use of nanoparticles (NPs) may present a solution for overcoming the limitations of the currently proposed methods of dye-based tumor delineation, due to their several advantages, such as high loading of drugs and contrast agents, nontoxicity of the matrix, and selective tumor targeting, including passive targeting by the enhanced permeability and retention (EPR) effect and active targeting by a surface- conjugated, tumor-specifi c targeting moiety. [ 11 ] Iron oxide- based NPs tagged with the near-infrared (NIR) fl uorescent dye Cy5.5 have been suggested as a method for dye-based intraoperative delineation of brain tumors. [ 12 ] However, one practical fl aw of Cy5.5-tagged NPs is that, despite the deep tissue penetrating capability of NIR fl uorescence, the NIR fl uorescence of Cy5.5 is invisible to the naked eye and can only be visualized on a separate monitor. Our group has pro- posed an alternative approach, based on the brain-tumor- targeted delivery of a visual delineating reagent made of NPs. [ 13 ] We performed a preliminary proof-of-principle study on visual tumor delineation by using NP surface-conjugated F3 peptides for glioma targeting, with polyacrylamide (PAA) NPs containing the blue dye, Coomassie Brilliant Blue G-250 (CB). [ 13 ] CB turned out to be a very good candidate as a vis- ible color contrast enhancer for intraoperative tumor deline- ation. [ 13 ] It has a vivid blue color in the pH 3 to 11 range, with a very high molar extinction coeffi cient. [ 14 ] It is also known to be safe for intravenous injection into the human body, even at very high doses. [ 15 ] However, our approach using the pre- viously reported CB-loaded NPs still had a signifi cant chal- lenge with respect to application in vivo. Because the NPs were prepared by loading CB into preformed blank NPs by “physical” adsorption (termed “post-loading”), they pro- duced a rather high degree of nonspecifi c cell staining with the passage of time ( Figure 1 ), [ 10 ] which may lead to ineffi - cient specifi c tumor staining in vivo. Herein, we report signifi cant improvements made to over- come the drawbacks of the preliminary approach, including: 1) development of a covalently linkable CB derivative to pre- vent unwanted premature dye leaching; 2) modifi cation of the NP synthesis procedures to achieve suffi cient dye loading by covalent linkage, and 3) introduction of polyethylene glycol (PEG)-containing crosslinkers for conjugation between www.small-journal.com © 2012 Wiley-VCH 9L-bearing rats with a cranial window, when compared with CB-post-loaded PAA NPs. The tumor-specifi c, nonleachable, blue-colored NPs do give good tumor delineation, which can be easily visualized by the naked eye, with enough contrast to potentially enable complete resection of the tumor with no extra equipment, no interruptions, and no unusual lighting conditions during surgery. 2. Results and Discussion 2.1. Synthesis of a Crosslinkable CB Derivative and its Characterization As a fi rst step to prepare CB covalently linked PAA NPs, commercially available CB was derivatized to an acrylamide- terminated form, by two-step reaction as summarized in Scheme 1 . The two sulfonic acid groups were converted into sulfonyl chloride groups, [ 16 ] forming an intermediate (CB-I), and were then changed into sulfamide groups after reacting with N -(3-aminopropyl)methacrylamide (APMA) under ba- sic conditions [ 17 ] to make CB-APMA. This modifi cation was NH3C NN O CH3 S Cl O O H3C CH3 S O Cl NH3C N H3C N H O CH3 S O- O O H3C CH3 S O O ONa i Verlag GmbH & Co. KGaA, Weinheim small 2012, DOI: 10.1002/smll.201101607 Hydrogel Nanoparticles with Covalently Linked Coomassie Blue performed without separation of CB-I, which is moisture sen- sitive, but the identity of CB-I was confi rmed by mass spec- trometry showing a molecular ion at m / z 868.1. The color of CB and CB-APMA is bright blue, whereas that of CB-I is green. The CB-APMA was obtained in a dried powder form, with 68% yield, and had 97% purity by elemental analysis (C 61 H 74 N 7 O 7 S 2 + (OH − ) 0.6 (SO 4 2 − ) 0.2 : C 64.04, H 6.58, N 8.58, O 11.75, S 6.17). The CB-APMA was characterized by 1 H NMR (1D and 2D), mass, and IR spectrometric measurements. The NMR proton assignments for CB-APMA are listed as follows: 1 H NMR (CD 2 Cl 2 , 500 MHz): δ = 7.91–7.69 (m, 4H; Ar − H), 7.47 (t, J = 7.5 Hz, 2H; CONH), 7.43–7.34 (m, 3H; Ar − H), 7.34–7.26 (m, 3H; Ar − H), 7.25–7.18 (m, 2H; Ar − H), 7.17–7.10 (m, 2H; Ar − H), 7.04 (t, J = 6.5 Hz, 2H; SO 2 − NH), 6.97–6.74 (m, 4H; Ar − H), 6.74–6.44 (m, 4H; Ar − H), 5.87–5.64 (m, 2H; CH 2 � ), 5.31–5.10 (m, 2H; CH 2 � ), 4.73(s, 4H; Ar − CH 2 ), 4.02 (q, J = 7 Hz, 2H; CH 3 − CH 2 −O), 3.85–3.49 (m, 4H; CH 3 −CH 2 − N), 3.49–3.41 (m, 4H; � CONH − CH 2 −), 2.99–2.71 (m, 4H; SO 2 NH � CH 2 ), 1.94–1.84 (m, 6H; CH 3 − C � ), 1.84–1.71 (br, 6H; CH 3 −Ar), 1.70–1.54 (m, 4H; CH 2 CH 2 CH 2 ), 1.38 (t, J = 7 Hz, 3H; CH 3 −CH 2 −O), 1.30 ppm (t, J = 7 Hz, 6H; CH 3 −CH 2 − N). The identity of CB-APMA was also confi rmed by the mass spectrum, which showed a molecular ion at m / z 1080.50. The IR (KBr) spectra confi rmed the presence of amide C � O at 1654, CO − NH at 1605, C � CH at 1494, and SO 2 −NH at 1166 cm − 1 . (More detailed analysis data are avail- able in the Supporting Information.) 2.2. Preparation of NPs Containing CB-APMA by Covalent Linkage CB covalently linked PAA NPs were prepared by reverse microemulsion polymerization with monomer mixtures con- taining CB-APMA, acrylamide (monomer), APMA (comon- omer), and glycerol dimethacrylate (GDMA, crosslinker; see Scheme S1, Supporting Information). CB-encapsulated and CB-post-loaded PAA NPs were also prepared for comparison (Scheme S1). The crosslinker, GDMA, contains hydrolyzable ester bonds, thus making the prepared NPs biodegradable in vivo. [ 11 , 18 ] APMA was included for providing the amine functionality to the NPs, so the NPs can be modifi ed with F3 peptides and PEG units (Scheme S2, Supporting Information). The CB-APMA is insoluble in water and can also serve as a crosslinker as it has two polymerizable carbon double bonds ( − C � C − ). The inclusion of CB-APMA was found to greatly infl uence the formation of the NPs. Thus, the synthetic proto- col for CB-linked PAA NPs had to be signifi cantly modifi ed from that of CB-encapsulated or CB-post-loaded PAA NPs. The monomers were dissolved in a DMF/water mixture. The amount (mole) of GDMA in the monomer mixture was re- duced by the added amount of CB-APMA, which kept the amount of the crosslinkers the same as that used for typical PAA NPs. The amount of surfactants was increased by a fac- tor of ≈ 3. Compared to preparing blank PAA NPs, about 16 times higher initiator quantities were required for the initia- tion of the polymerization, probably because of the signifi cant quenching of the produced radicals by the larger amounts of dye and surfactant. © 2012 Wiley-VCH Verlag Gmbsmall 2012, DOI: 10.1002/smll.201101607 2.3. Surface Modifi cations and Cancer-Specifi c Targeting To achieve brain-cancer-specifi c targeted delivery of CB- loaded PAA NPs, two surface modifi cations/conjugations were made. First, the surface of CB-loaded NPs was PEGylated (coated with polyethylene glycol) by using the heterobi- functional PEG, SCM-PEG-MAL, as a crosslinking reagent between the NP and a targeting moiety. Its amine-reactive terminal (succinimidyl carboxymethyl ester, SCM) chemically links to the primary amine group present on the surface of PAA NPs. Then, the sulfhydryl-reactive terminal (maleimidyl ester, MAL) binds to the sulfhydryl group in F3-Cys peptide for cancer targeting or to l -cysteine for control (Scheme S2). The surface charge in each modifi cation step was measured as an indicator showing whether the product was formed as de- sired, using the dynamic light scattering (DLS) zeta potential measurement technique. Unmodifi ed NPs exhibit a relatively strong positive charge ( + 17.38 ± 3.30 mV in zeta potential) due to the presence of protonated amine groups (-NH 3 + ) on the surface. PEGylation on the surface resulted in a signifi cantly reduced positive charge ( + 2.98 ± 1.40 mV). The coverage of PAA NPs by PEG was estimated to be about 40 PEG mol- ecules per single PAA NP by UV/Vis absorption-based analy- sis done after treating the NPs with fl uorescein-labeled PEG- succinimidyl ester. F3-conjugated NPs regained a net positive surface charge ( + 15.11 ± 2.36 mV) because of the strong basic amino acid composition of the F3 peptide. The introduction of PEG in between the NP and the F3 peptide was another effort to improve in vivo tumor delinea- tion effi cacy, compared to the previous approach that utilized a relatively small molecular crosslinker, sulfo-SMCC. [ 13 ] Surface PEGylation can effectively suppress the nonspecifi c binding by the free motion of the electrically neutral, long polymer PEG chains, thereby allowing only target-specifi c binding to the cancer cells by the F3 peptide. Also, PEGyla- tion can improve the colloidal stability (suspendability) of the NPs in physiological media, which results in longer plasma circulation times. Such effects should improve the in vivo function of the F3-targeted CB-linked PAA NPs. 2.4. NP Characterization The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images show that the CB-linked NPs have spherical particulate morphology and a size of ≈ 45 nm in diameter, whereas the CB-encapsulated or CB-post-load- ed NPs have a size of ≈ 30 nm ( Figure 2 ). The average sizes (diameters) of CB-linked PAA NPs, CB-encapsulated PAA NPs, and CB-post-loaded PAA NPs in water, measured by DLS, are (86.8 ± 21.0), (57.3 ± 11.8) and (54.1 ± 10.2) nm, re- spectively, whereas for blank PAA NPs it is 53.4 ± 9.5 nm. The fact that the hydrodynamic sizes of these NPs are much larger than those determined by the electron micrographs indicates that the PAA NPs can swell in aqueous solution, manifesting a hydrogel characteristic. The average sizes of the F3-targeted and nontargeted (PEGylated) CB-linked PAA NPs are, respectively, (90.3 ± 7.1) and (88.6 ± 11.9) nm by DLS, which indicates that surface conjugation does not affect the particle 3www.small-journal.comH & Co. KGaA, Weinheim G. Nie et al.full papers Figure 2 . SEM and TEM images of the prepared NPs: A) CB covalently linked PAA NPs; B) CB-encapsulated PAA NPs; and C) CB-post-loaded PAA NPs. 4 www.small-journal.com © 2012 Wiley-VCH V size signifi cantly. The size of all CB-loaded PAA NPs, before and after surface modifi cation, falls into the range of 10–100 nm, which is generally the accepted optimal size range for in vivo applications, because a particle in this range is too large to undergo renal elimination and too small to be recognized by phagocytes. [ 19–21 ] We also confi rmed that various dye loading methods (post-loading and covalent linking) did not make recogniz- able differences in the zeta potential value. The amount of conjugated F3 peptide was determined to be 0.027 μ mol mg − 1 of NP by weight, by quantitative amino acid assays. We believe that F3 peptides are conjugated mostly to the surface through PEG crosslinker because of: 1) PEG molecules being mostly located on the surface, as shown by the signifi cant sur- face charge reduction after PEGylation and the regaining of positive surface charge after F3 conjugation (Section 2.3); and 2) considering the relatively large molecular weight of the F3 peptide (3566 g mol − 1 ). Our previous studies show that mol- ecules having molecular weights larger than ≈ 2500 Da do not leach out of PAA NPs, thus indicating that such molecules can hardly enter into the NPs from the outside. [ 22 ] 2.5. Quantifi cation of Dye Loading Effi ciency The absorption spectra of CB-linked NPs in aqueous solution show that their absorption maximum is observed at 610 nm while that of CB-free dye in aqueous solution is at 597 nm (Supporting Information). Just as for the solvent-dependent spectral shifts of the dyes, [ 23 ] such a peak shift may be due to a polarity and polarizability of the CB-linked NP matrix that differs from that of water or that of the normal PAA NP matrix. CB has a peak at 618 nm when dissolved in DMF. The observed peak shift does not produce signifi cant visible color changes (Supporting Information). The loading of CB per NP is an important factor for suc- cessful visible color-based tumor delineation. In our studies with CB-free dye, the estimated dose for obtaining a suf- fi cient color delineation effect in a rat brain tumor window (BTW) model was about 70 mg kg − 1 , although the color con- trast started to fade at about 60 min after injection. [ 9 ] The required CB dose for the CB-loaded NPs may be lower than the CB-free dye dose, through the expected enhancement in effectivity due to passive delivery by the EPR effect, the surface-conjugated PEG, and the tumor-specifi c targeting moieties. We aimed at using half of the free CB dose, that is, 35 mg kg − 1 , for CB-loaded NPs. According to our previous in vivo animal studies, the PAA NPs showed no evidence of alterations in histopathology or clinical chemistry values at doses of 10 mg kg − 1 to 1 g kg − 1 . [ 11 ] We chose the maximum NP dose to be equal to or less than 500 mg kg − 1 . To match the dose require