RESUMO
Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.
Assuntos
Técnicas Biossensoriais , Proteínas Luminescentes/isolamento & purificação , Ficocianina/química , Trichodesmium/metabolismo , Biliverdina/química , Ciclo Celular/fisiologia , Escherichia coli/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/efeitos da radiação , Mutação , Ficocianina/metabolismo , Conformação Proteica , Estabilidade Proteica , Subunidades Proteicas , Proteína Vermelha FluorescenteRESUMO
Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41's binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue-NP41-bound nerves, followed by biotin hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10-7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve "ghosts" months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.
RESUMO
New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [(18)F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [(18)F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases.
Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Radioisótopos de Flúor , Tomografia por Emissão de Pósitrons/métodos , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Cetuximab/metabolismo , Humanos , Camundongos , Imagem Óptica , Radioquímica , Estreptavidina/metabolismoRESUMO
In real time: thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Trombina/metabolismo , Animais , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Camundongos , Camundongos TransgênicosRESUMO
We recently developed activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix metalloproteinase activity, such as tumors. Here we use parallel in vivo and in vitro selection with phage display to identify novel tumor-homing ACPPs with no bias for primary sequence or target protease. Specifically, phage displaying a library of ACPPs were either injected into tumor-bearing mice, followed by isolation of cleaved phage from dissected tumor, or isolated based on selective cleavage by extracts of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts. The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast. This uniquely identified ACPP was not cleaved by matrix metalloproteinases or various coagulation factors but was efficiently cleaved by plasmin and elastases, both of which have been shown to be aberrantly overexpressed in tumors. The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Neoplasias/metabolismo , Peptídeo Hidrolases/metabolismo , Biblioteca de Peptídeos , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Extratos de TecidosRESUMO
We combine a novel boronate trap for F(-) with a near-infrared fluorophore into a single molecule. Attachment to targeting ligands enables localization by positron emission tomography (PET) and near-infrared fluorescence (NIRF). Our first application of this generic tag is to label Lymphoseek (tilmanocept), an agent designed for receptor-specific sentinel lymph node (SLN) mapping. The new conjugate incorporates (18)F(-) in a single, aqueous step, targets mouse SLN rapidly (1 h) with reduced distal lymph node accumulation, permits PET or scintigraphic imaging of SLN, and enables NIRF-guided excision and histological verification even after (18)F decay. This embodiment is superior to current SLN mapping agents such as nontargeted [(99m)Tc]sulfur colloids and Isosulfan Blue, as well as the phase III targeted ligand [(99m)Tc]SPECT Lymphoseek counterpart, species that are visible by SPECT or visible absorbance separately. Facile incorporation of (18)F into a NIRF probe should promote many synergistic PET and NIRF combinations.
Assuntos
Corantes Fluorescentes , Raios Infravermelhos , Marcação por Isótopo/métodos , Linfonodos/metabolismo , Imagem Molecular/métodos , Fenômenos Ópticos , Tomografia por Emissão de Pósitrons/métodos , Animais , Ácidos Borônicos/química , Ácidos Borônicos/farmacocinética , Dextranos/química , Dextranos/farmacocinética , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Radioisótopos de Flúor , Cinética , Linfonodos/diagnóstico por imagem , Linfonodos/cirurgia , Mananas/química , Mananas/farmacocinética , Camundongos , Compostos de Organotecnécio/química , Compostos de Organotecnécio/farmacocinética , Ácido Pentético/química , Ácido Pentético/farmacocinética , Radioquímica , Biópsia de Linfonodo Sentinela , Pentetato de Tecnécio Tc 99m/análogos & derivados , Tomografia Computadorizada por Raios XRESUMO
Patients with advanced cancers are treated with combined radiotherapy and chemotherapy, however curability is poor and treatment side effects severe. Drugs sensitizing tumors to radiotherapy have been developed to improve cell kill, but tumor specificity remains challenging. To achieve tumor selectivity of small molecule radiosensitizers, we tested as a strategy active tumor targeting using peptide-based drug conjugates. We attached an inhibitor of the DNA damage response to antibody or cell penetrating peptides. Antibody drug conjugates honed in on tumor overexpressed cell surface receptors with high specificity but lacked efficacy when conjugated to the DNA damage checkpoint kinase inhibitor AZD7762. As an alternative approach, we synthesized activatable cell penetrating peptide scaffolds that accumulated within tumors based on matrix metalloproteinase cleavage. While matrix metalloproteinases are integral to tumor progression, they have proven therapeutically elusive. We harnessed these pro-tumorigenic extracellular proteases to spatially guide radiosensitizer drug delivery using cleavable activatable cell penetrating peptides. Here, we tested the potential of these two drug delivery platforms targeting distinct tumor compartments in combination with radiotherapy and demonstrate the advantages of protease triggered cell penetrating peptide scaffolds over antibody drug conjugates to deliver small molecule amine radiosensitizers.
Assuntos
Peptídeos Penetradores de Células , Radiossensibilizantes , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Peptídeo HidrolasesRESUMO
Matrix metalloproteinases-2 and -9 (MMP-2/-9) are key tissue remodeling enzymes that have multiple overlapping activities critical for wound healing and tumor progression in vivo. To overcome issues of redundancy in studying their functions in vivo, we created MMP-2/-9 double knockout (DKO) mice in the C57BL/6 background to examine wound healing. We then bred the DKO mice into the polyomavirus middle T (PyVmT) model of breast cancer to analyze the role of these enzymes in tumorigenesis. Breeding analyses indicated that significantly fewer DKO mice were born than predicted by Mendelian genetics and weaned DKO mice were growth compromised compared with wild type (WT) cohorts. Epithelial wound healing was dramatically delayed in adult DKO mice and when the DKO was combined with the PyVmT oncogene, we found that the biologically related process of mammary tumorigenesis was inhibited in a site-specific manner. To further examine the role of MMP-2/-9 in tumor progression, tumor cells derived from WT or DKO PyVmT transgenic tumors were grown in WT or DKO mice. Ratiometric activatable cell penetrating peptides (RACPPs) previously used to image cancer based on MMP-2/-9 activity were used to understand differences in MMP activity in WT or knockout syngeneic tumors in WT and KO animals. Analysis of an MMP-2 selective RACPP in WT or DKO mice bearing WT and DKO PyVmT tumor cells indicated that the genotype of the tumor cells was more important than the host stromal genotype in promoting MMP-2/-9 activity in the tumors in this model system. Additional complexities were revealed as the recruitment of host macrophages by the tumor cells was found to be the source of the tumor MMP-2/-9 activity and it is evident that MMP-2/-9 from both host and tumor is required for maximum signal using RACPP imaging for detection. We conclude that in the PyVmT model, the majority of MMP-2/-9 activity in mammary tumors is associated with host macrophages recruited into the tumor rather than that produced by the tumor cells themselves. Thus therapies that target tumor-associated macrophage functions have the potential to slow tumor progression.
Assuntos
Carcinogênese/metabolismo , Peptídeos Penetradores de Células/metabolismo , Neoplasias Mamárias Animais/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Cicatrização , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Feminino , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
A fundamental goal of many surgeries is nerve preservation, as inadvertent injury can lead to patient morbidity including numbness, pain, localized paralysis and incontinence. Nerve identification during surgery relies on multiple parameters including anatomy, texture, color and relationship to surrounding structures using white light illumination. We propose that fluorescent labeling of nerves can enhance the contrast between nerves and adjacent tissue during surgery which may lead to improved outcomes. Methods: Nerve binding peptide sequences including HNP401 were identified by phage display using selective binding to dissected nerve tissue. Peptide dye conjugates including FAM-HNP401 and structural variants were synthesized and screened for nerve binding after topical application on fresh rodent and human tissue and in-vivo after systemic IV administration into both mice and rats. Nerve to muscle contrast was quantified by measuring fluorescent intensity after topical or systemic administration of peptide dye conjugate. Results: Peptide dye conjugate FAM-HNP401 showed selective binding to human sural nerve with 10.9x fluorescence signal intensity (1374.44 ± 425.96) compared to a previously identified peptide FAM-NP41 (126.17 ± 61.03). FAM-HNP401 showed nerve-to-muscle contrast of 3.03 ± 0.57. FAM-HNP401 binds and highlight multiple human peripheral nerves including lower leg sural, upper arm medial antebrachial as well as autonomic nerves isolated from human prostate. Conclusion: Phage display has identified a novel peptide that selectively binds to ex-vivo human nerves and in-vivo using rodent models. FAM-HNP401 or an optimized variant could be translated for use in a clinical setting for intraoperative identification of human nerves to improve visualization and potentially decrease the incidence of intra-surgical nerve injury.
Assuntos
Imagem Molecular/métodos , Neuroimagem/métodos , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Cirurgia Assistida por Computador/métodos , Administração Intravenosa , Animais , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/metabolismo , Humanos , Camundongos , Peptídeos/administração & dosagem , Peptídeos/metabolismo , Ligação Proteica , RatosRESUMO
Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Maitansina/análogos & derivados , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Radiossensibilizantes/farmacologia , Trastuzumab/farmacologia , Moduladores de Tubulina/farmacologia , Ado-Trastuzumab Emtansina , Aminobenzoatos/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Receptores ErbB/imunologia , Feminino , Humanos , Maitansina/farmacologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Oligopeptídeos/farmacologia , Radiação Ionizante , Transdução de SinaisRESUMO
Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides.
Assuntos
Oligopeptídeos/farmacologia , Radiossensibilizantes/farmacologia , Animais , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Peptídeos Penetradores de Células/administração & dosagem , Quimiorradioterapia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Quebras de DNA de Cadeia Dupla , Sistemas de Liberação de Medicamentos , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Oligopeptídeos/administração & dosagem , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Tolerância a Radiação , Radiossensibilizantes/administração & dosagem , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Activatable cell-penetrating peptides (ACPP) provide a general strategy for molecular targeting by exploiting the extracellular protease activities associated with disease. Previous work used a matrix metalloproteinase (MMP-2 and 9)-cleavable sequence in the ACPP to target contrast agents for tumor imaging and fluorescence-guided surgery. To improve specificity and sensitivity for MMP-2, an integrin α(v)ß(3)-binding domain, cyclic-RGD, was covalently linked to the ACPP. This co-targeting strategy relies on the interaction of MMP-2 with integrin α(v)ß(3), which are known to associate via the hemopexin domain of MMP-2. In U87MG glioblastoma cells in culture, dual targeting greatly improved ACPP uptake compared with either MMP or integrin α(v)ß(3) targeting alone. In vivo, dual-targeted ACPP treatment resulted in tumor contrast of 7.8 ± 1.6, a 10-fold higher tumor fluorescence compared with the negative control peptide, and increased probe penetration into the core of MDA-MB-231 tumors. This platform also significantly improved efficacy of the chemotherapeutic monomethylauristatin E (MMAE) in both MDA-MB-231 orthotopic human and syngeneic Py230 murine breast tumors. Treatment with cyclic-RGD-PLGC(Me)AG-MMAE-ACPP resulted in complete tumor regression in one quarter of MDA-MB-231 tumor-bearing mice, compared with no survival in the control groups. This rational mechanism for amplified delivery of imaging and potent chemotherapeutic agents avoids the use of antibodies and may be of considerable generality.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Glioblastoma/diagnóstico por imagem , Integrina alfaVbeta3/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Animais , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Meios de Contraste/química , Meios de Contraste/metabolismo , Sistemas de Liberação de Medicamentos , Glioblastoma/patologia , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/genética , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/genética , Camundongos , Terapia de Alvo Molecular , Tomografia por Emissão de Pósitrons , RadiografiaRESUMO
Thrombin and other coagulation enzymes have been shown to be important during atherosclerotic disease development. Study of these proteases is currently limited because of lack of robust molecular imaging agents for imaging protease activity in vivo. Activatable cell penetrating peptides (ACPPs) have been used to monitor MMP activity in tumors and, in principle, can be modified to detect other proteases. We have developed a probe that incorporates the peptide sequence DPRSFL from the proteinase activated receptor 1 (PAR-1) into an ACPP and shown that it is preferentially cleaved by purified thrombin. Active thrombin in serum cleaves DPRSFL-ACPP with >90% inhibition by lepirudin or argatroban. The DPRSFL-ACPP cleavage product accumulated in advanced atherosclerotic lesions in living mice, with 85% reduction in retention upon pre-injection of mice with hirudin. Uptake of the ACPP cleavage product was highest in plaques with histological features associated with more severe disease. Freshly resected human atheromas bathed in DPRSFL-ACPP retained 63% greater cleavage product compared to control ACPP. In conclusion, DPRSFL-ACPP can be used to study thrombin activity in coagulation and atherosclerosis with good spatial and temporal resolution. Thrombin-sensitive ACPPs may be developed into probes for early detection and intraoperative imaging of high risk atherosclerotic plaques.
Assuntos
Aorta/metabolismo , Peptídeos Penetradores de Células/farmacocinética , Corantes Fluorescentes/farmacocinética , Placa Aterosclerótica/metabolismo , Receptor PAR-1/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Animais , Antitrombinas/farmacologia , Aorta/enzimologia , Aorta/patologia , Arginina/análogos & derivados , Hirudinas/farmacologia , Histocitoquímica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Ácidos Pipecólicos/farmacologia , Placa Aterosclerótica/enzimologia , Placa Aterosclerótica/patologia , Proteínas Recombinantes/farmacologia , Sulfonamidas , Trombina/antagonistas & inibidoresRESUMO
OBJECTIVES/HYPOTHESIS: By phage display, we have developed a novel peptide (NP41) that binds selectively to nerves following systemic administration. We evaluated the pattern of facial nerve labeling with fluorescently-labeled NP41 (F-NP41). We also tested whether F-NP41 highlights facial nerves well enough to identify nerve stumps accurately several weeks after nerve transection. STUDY DESIGN: Forty-seven wild-type mice were studied prospectively. One surgeon performed the nerve transection, reanastomosis, and monitoring of functional recovery. METHODS: Fluorescent labeling: F-NP41 was administered intravenously (20 mice). Nerve labeling was studied with fluorescence microscopy. Transection and reanastomosis: the right facial nerve was transected (25 mice). Three weeks after transection, F-NP41 was administered intravenously and fluorescence microscopy was used to identify the nerve stumps and reanastomosis in one group. Nerve identification and reanastomosis was performed with white light in another group without F-NP41. The control group underwent sham surgery. Time to nerve identification was recorded. Functional recovery was monitored for at least 8 weeks. RESULTS: We found excellent labeling of intact and transected facial nerves following F-NP41 administration. Several weeks following nerve transection, F-NP41 provided accurate identification of the proximal and distal nerve stumps. Following reanastomosis, time to recovery and level of functional recovery was similar in the absence and presence of F-NP41. CONCLUSIONS: We show improved visualization of facial nerves with a novel systemically applied fluorescently labeled probe. Use of F-NP41 resulted in accurate identification of facial nerve stumps several weeks following transection. Functional recovery was similar with and without the use of F-NP41.
Assuntos
Traumatismos do Nervo Facial/patologia , Nervo Facial/patologia , Fluoresceínas , Corantes Fluorescentes , Microscopia de Fluorescência , Biblioteca de Peptídeos , Peptídeos , Anastomose Cirúrgica , Animais , Proteínas de Bactérias/genética , Nervo Facial/cirurgia , Traumatismos do Nervo Facial/cirurgia , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Degeneração Neural/patologia , Regeneração Nervosa/fisiologia , Antígenos Thy-1/genéticaRESUMO
Nerve preservation is an important goal during surgery because accidental transection or injury leads to significant morbidity, including numbness, pain, weakness or paralysis. Nerves are usually identified by their appearance and relationship to nearby structures or detected by local electrical stimulation (electromyography), but thin or buried nerves are sometimes overlooked. Here, we use phage display to select a peptide that binds preferentially to nerves. After systemic injection of a fluorescently labeled version of the peptide in mice, all peripheral nerves are clearly delineated within 2 h. Contrast between nerve and adjacent tissue is up to tenfold, and useful contrast lasts up to 8 h. No changes in behavior or activity are observed after treatment, indicating a lack of obvious toxicity. The fluorescent probe also labels nerves in human tissue samples. Fluorescence highlighting is independent of axonal integrity, suggesting that the probe could facilitate surgical repair of injured nerves and help prevent accidental transection.