Bioconjugate Chemistry

Ultrasound technology is a powerful tool for medical imaging and diagnosis especially when used in conjunction with a contrast agent to target proteins of interest. However, current ultrasound contrast agents are lacking the combination of specificity, stability, and size optimization. In this study we introduce and confirm the feasibility of using nanometer sized gas vesicles (GV) chemically click conjugated to antibodies (mAb) to make the first site-specific mAb-GV conjugates as a durable cancer cell targeting ultrasound molecular contrast agents. Protein expression of human epidermal growth factor receptor 2 (HER2) and programmed death-ligand 1 (PD-L1) were tested along with the antibody targeting efficiency using cancer cell lines and primary cells isolated from tumor bearing mice. The mAb conjugation was optimized to a site-specific method using the mAb glycans and tested with the addition of a clinically used mAb to target trophoblast cell surface antigen 2 (Trop-2). The developed contrast agents utilize the stability of GVs and the specificity of antibodies to label cancer biomarkers in various types of tumors for ultrasound imaging.

Detecting protein-protein interactions within cells is challenging. Transgenic approaches risk altering protein function via fluorescent tagging, while in situ methods lack in vivo compatibility. Here, we introduce fluorogenic probes with dual-tetrazine pegylated branched arms linked to xanthene dye. Activation requires both tetrazine arms to interact simultaneously with target proteins, enabling dual-substrate recognition. We applied our method to detect protein-protein interactions in both fixed and living cells, utilizing antibody conjugation for fixed cells and genetic code expansion for real-time detection in living cells. Our strategy ensures versatile applicability and seamless transition between fixed and living systems.

TLR 7/8 agonists are highly potent immunostimulators, though their clinical translation has been met with mixed success, due to their high toxicity as a result of an unregulated systemic immune activation. There is enormous potential to augment cancer immunotherapies with synthetic TLR 7/8 agonists, though a thorough control of pharmacokinetics and localization is needed for the general use of TLR 7/8 agonists in cancer immunotherapy. Herein, we control localization of TLR 7/8 agonists, by exploiting the extensive tissue retention of poly(acrylic acid-co-styrene). In a murine CT26 model, we find that covalently attaching TLR 7/8 agonists to the copolymer allows for retaining the drug in the tumor microenvironment for at least 15 weeks, after intratumoral injection, and results in a curative monotherapy. The copolymer itself is a new avenue for attaining prolonged tissue rentention for covalently attached drugs.

Monoclonal antibodies have had a remarkable impact on cancer therapy due to their high target specificity. However, their large molecular weight results in slow blood clearance, which can take weeks to clear from circulation. As companion nuclear imaging and diagnostic tools, these characteristics force delayed imaging and the use of isotopes with long half-lives such as 89Zr. For optimal clinical application, it is desirable that radioimmunoconjugates remain in the blood for just enough time to accumulate adequately in target tissues, while non-targeted or circulating radioactivity is ideally rapidly excreted from the body to maximize imaging contrast and minimize radiation dose to healthy tissues. We addressed the current challenges of antibody-based imaging by developing rituximab radioimmunoconjugates that accumulate sufficient activity for tumor imaging within 24 h of administration, while clearing circulating radioactivity via administration of a small molecule clearing agent. Rituximab, an anti-CD20 monoclonal antibody, is used as standard first-line therapy for diffuse large B-cell lymphoma. CD20 is expressed by 95% of B-lymphocytes and their malignant counterparts, making it a therapeutic target for B-cell malignancies. We attached 125I, 68Ga, and 89Zr to rituximab using a "clickable" linker containing trans-cyclooctene and tested the ability of tetrazines to induce the inverse electron demand Diels-Alder reaction (iEDDA) after antibody administration. This "tetrazine-knock-out" (TKO) approach liberates the radioactivity from rituximab in the bloodstream, resulting in its rapid renal excretion which enhances target-to-background ratios, and minimizes off-target radiation exposure. Due to the internalization of the radioimmunoconjugate in CD20+ tumor cells, no substantial clearance was observed from Raji xenografts. We characterized different leaving groups, several cellular models and antibodies with distinct internalizaing properties. The TKO approach opens opportunities to use radiolabeled antibodies for low-abundance or heterogeneously expressed biologic targets and may allow radioimmunotherapy (RIT) for targets traditionally untenable due to dose-limiting toxicities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/596510v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11bc2f0org.highwire.dtl.DTLVardef@19b4f72org.highwire.dtl.DTLVardef@17cee32org.highwire.dtl.DTLVardef@fa4491_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hydrolases are important molecules that are involved in a wide range of biological functions and their activities are tightly regulated in healthy or diseased states. Detecting or imaging the activities of hydrolases, therefore, can reveal underlying molecular mechanisms in the context of cells to organisms, and their correlation with different physiological conditions can therefore be used in diagnosis. Due to the nature of hydrolases, substrate-based probes can be activated in their catalytic cycles, and cleavage of covalent bonds frees reporter moieties. For test-tube type bulk detection, spatial resolution is not a measure of importance, but for cell- or organism-based detection or imaging, spatial resolution is a key factor for probe sensitivity that influences signal-to-background ratio. One strategy to improve spatial resolution of the probes is to form a covalent linkage between the reporter moiety and intracellular proteins upon probe activation by the enzyme. In this work, we developed a generalizable linker chemistry that would allow in situ labeling of various imaging moieties via quinone methide species. To do so, we synthesized probes containing a monofluoromethyl or a difluoromethyl groups for {beta}-galactosidase activation, while using fluorescein as a fluorescent reporter. The labeling efficacy of these two probes was evaluated in vitro. The probe bearing a monofluormethyl group exhibited superior labeling efficiency in imaging {beta}-galactosidase activity in living cells. This study provides a versatile linker for applying quinone methide chemistry in the development of hydrolase-targeting probes involving in situ labeling.

Aptamers are oligonucleotides with antibody-like binding function, selected from large combinatorial libraries. In this study, we modified a DNA aptamer library with N-hydroxysuccinimide esters, enabling covalent reactivity with cognate proteins. We selected for the ability to bind to mouse monoclonal antibodies, resulting in the isolation of two distinct covalent binding motifs. The covalent aptamers are specific for the Fc region of mouse monoclonal IgG1 and are cross-reactive with mouse IgG2a and other IgGs. Investigation into the covalent reactivity of the aptamers revealed a dependence on micromolar concentrations of Cu2+ ions which can be explained by residual catalyst remaining after modification of the aptamer library. The aptamers were successfully used as adapters in the formation of antibody-oligonucleotide conjugates (AOCs) for use in detection of HIV protein p24 and super-resolution imaging of actin. This work introduces a new method for the site-specific modification of native monoclonal antibodies and may be useful in applications requiring AOCs or other antibody conjugates.

Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with the hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide 68Ga. [68Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [68Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [68Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 {+/-} 0.58 %ID g-1 (n = 3; 2 h post-administration) and was reduced by [~]50% in the blocking group (2.47 {+/-} 0.42 %ID g-1; n = 3; P-value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.

Antibody conjugates play a central role across multiple healthcare sectors with a prime example being antibody-drug conjugates (ADCs). Although widely used lysine and hinge cysteine conjugation methods yield products, the lack of site-specificity and spatial control along with the highly heterogeneous composition are significant limitations. We describe a facile supramolecular assembly method based on heterodimer coiled-coil formation for site-specific antibody conjugation. The method affords uniform loading of diverse payloads including anti-cancer agents, polymers, enzymes, fluorophores, etc. under mild aqueous conditions. Further, the facile convergent approach capitalizes on the independent strengths and flexibility of protein expression and peptide chemistry culminating in a final self-assembly step. Coiled-coil conjugation perseveres both antibody antigen binding sites for target engagement and heavy chains constant domains for Fc binding and recycling. An ADC loaded with monomethyl auristatin E targeting HER2+ tumors significantly reduces tumor volume in a human ovarian cancer xenograft model outperforming the antibody alone with validated performance against a best-in-class therapeutic. Supramolecular assembly-driven bioconjugation expands the bioorthogonal chemistry toolbox for antibody modification and opens new avenues for advanced antibody conjugates with multiple payloads.

Nucleic acid crosslinkers that covalently join commentary strands have applications as both pharmaceuticals and biochemical probes. Psoralen is a popular crosslinker moiety that reacts with double stranded DNA and RNA upon irradiation with long wave UV light. A commercially available compound EZ-Link Psoralen-PEG3-Biotin has been used in many studies to crosslink DNA and double strand RNA for genome-wide investigations. Here we present a novel probe, AP3B, which uses a psoralen derivative, 4-aminomethyltrioxsalen, to biotinylate nucleic acids. We show that this compound is 8-fold more effective at labeling DNA in cells and several hundred-fold more effective at crosslinking two strands of DNA in vitro than the commercially available compound EZ-Link Psoralen-PEG3-Biotin.

Single domain antibodies, often known as nanobodies, are versatile molecules with therapeutic and diagnostic applications, but they are primarily developed through immunization of camelids. This approach is not scalable by automation, not effective for non-immunogenic or toxic antigens, and prevents the use of modified scaffolds for altered pharmacokinetic properties. Synthetic libraries allow for pre-selection of a single domain framework tailored to its intended downstream use. One area of interest for these biologic vectors is radiopharmaceuticals. Ideal radiopharmaceutical pharmacokinetic properties differ from most traditional therapeutics, as short plasma circulation and rapid kidney clearance are necessary to avoid dose-limiting organ radiation. Although there are a growing number of nanobody radiopharmaceuticals in clinical trials, their frameworks and corresponding pharmacokinetic properties vary. One potential method for improving the development of novel single domain antibody radiopharmaceuticals is through synthetic libraries based on nanobodies with proven clinically acceptable pharmacokinetics. We developed a modular synthetic nanobody phage display vector based on the scaffold of the 2Rs15d nanobody that allows for manipulation of the binding and framework regions. Using this vector, we created a library of nanobodies with a randomized CDR2 containing over 1.7x106 unique sequences/{micro}L. As a proof-of-concept, we panned the library for nanobodies binding calreticulin (CALR), a protein critical in immunogenic cell death. One isolated clone, Cal3, has a measured affinity of 140 nM for CALR and is cross-reactive with mouse and human CALR. Using positron emission tomography (PET) imaging, the radiolabeled 64Cu-NOTA-Cal3 demonstrated CALR binding in vivo, representing the first reported synthetic nanobody characterized by PET imaging. This study demonstrates the feasibility of building and panning synthetic libraries for high-affinity radiopharmaceutical nanobodies as an alternative to immunized camelid libraries.

Recently, our lab developed GlycoDelete, a technology suite that allows a radical simplification of eukaryotic N-glycosylation. The technology allows to produce glycoproteins that carry single GlcNAc, LacNAc, or LacNAc-Sia type glycans on their N-linked glycosylation sequons. GlycoDelete-type N-glycans are uniquely suited for glycan-based conjugation purposes, as these provide a short, homogeneous and hydrophilic link to the protein backbone. Targeting GlycoDelete-glycans allows for highly site-specific conjugation at sites in the protein which are normally occupied by bulky glycans, thus ensuring minimal interference with protein structure and function. The current manuscript describes the evaluation and optimization of both chemical and chemo-enzymatic conjugation of molecules onto the GlycoDelete-type glycans of a limited set of benchmark proteins.

With the recent success of cell-based therapies, there has been a rapidly emerging interest in the engineering of cell-cell interactions and communications. Inspired by the natural intercellular material transfer process of trans-endocytosis or trogocytosis, we proposed that targeted farnesylated chemically self-assembled nanorings (farnesyl-CSANs) could serve as a biomimetic trogocytosis vehicle for engineering directional cargo transfer between cells; thus, allowing cell-cell interactions to be monitored, as well as facilitating communication between the cells by delivery of bioactive species. The membranes of sender cells were stably modified by hydrophobic insertion with the targeted farnesyl-CSANs and to be efficiently transferred to receiver cells expressing the appropriate receptor by endocytosis. CSAN-assisted cell-cell cargo transfer (C4T) was demonstrated to be receptor-specific and dependent on direct cell-cell interactions, the rate of receptor internalization and the amount of receptor expression. In addition, C4T was shown to facilitate cell-to-cell delivery of an apoptosis inducing drug, as wells as antisense oligonucleotides (ASO). Taken together, the C4T approach is a potentially versatile biomimetic trogocytosis platform that can be used to monitor cell-cell interactions, as well as the engineering of cell-cell communications, such as cell-based drug delivery.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality in men and women. Timely detection and diagnosis are key to management of CRC, which is under-diagnosed because colorectal aberrant crypt foci, hyperplastic polyps, and microadenomas are often missed with conventional colonoscopy. The enzyme cyclooxygenase-2 (COX-2) is overexpressed in early stages of colorectal carcinogenesis and plays an important regulatory role in the process, suggesting that it could be a valuable target for enhanced imaging of nascent disease. Thus, we have generated an alpaca-derived library of 73 COX-2-specific nanobody clones. Here, we describe one such nanobody, F9-K45Q-K77Q-ROX, in which two native lysine residues have been mutated followed by conjugation to a fluorophore at the N-terminus with retention of COX-2-selective binding. The site of fluorophore conjugation and COX-2 binding affinity of F9-K45Q-K77Q-ROX were determined by proteomic and microscale thermophoretic analyses, respectively. In cell culture studies using 1483 human head and neck squamous cell carcinoma cells, F9-K45Q-K77Q-ROX accumulated inside cells and bound to intracellular COX-2, as visualized by fluorescence microscopy. In vivo pharmacokinetic, and toxicological analyses revealed that F9-K45Q-K77Q-ROX is detectable in circulation with a plasma half-life of 17.9 min and there is no short-term toxicity associated with single injections of 10 mg/kg, 20 mg/kg, or 40 mg/kg doses at 24 h post-administration. Noninvasive in vivo fluorescence endoscopic imaging validated tumor-specific accumulation of F9-K45Q-K77Q-ROX in azoxymethane/dextran sodium sulfate-induced colorectal adenomas in mice. This work demonstrates the first COX-2-targeted nanobodies including a fluorescent derivative that offers significant promise for targeted endoscopic imaging of COX-2-expressing neoplasms. Significance StatementCurrent colorectal cancer screening procedures, such as white-light colonoscopy, chromoendoscopy, and narrow-band imaging aim to detect solid colon tumors and precursor lesions. However, these methods tend to detect only raised solid tumors and mature cancers, whereas precursor lesions, such as aberrant crypt foci, hyperplastic polyps, and small adenomas are frequently missed. To address the need for better visualization of early lesions, we developed a library of alpaca-derived nanobodies targeted to cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in colorectal adenomas. COX-2-targeted nanobodies bearing a fluorescent tag accumulate and are retained in colonic adenomas, facilitating their endoscopic visualization. This novel COX-2-targeted nanobody platform may also be valuable for early detection of other neoplastic diseases in which COX-2 overexpression occurs. (Word counts 119, limit 120)

siRNAs have become one of the most promising therapeutic agents because of their specificity and their potential to modulate the expression of gene-related diseases. Any gene of interest can be potentially up or down-regulated, making RNA-based technology the healthcare breakthrough of our era. However, the functional and specific delivery of siRNAs into tissues of interest and into the cytosol of target cells remains highly challenging, mainly due to the lack of efficient and selective delivery systems. Among the variety of carriers for siRNA delivery, peptides have become essential candidates because of their high selectivity, stability and conjugation versatility. Here, we describe the development of molecules encompassing siRNAs against SOD1, conjugated to peptides that target the LDLR, and their biological evaluation both in vitro and in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/526778v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@11146borg.highwire.dtl.DTLVardef@1537ee2org.highwire.dtl.DTLVardef@af67b5org.highwire.dtl.DTLVardef@1c1d7ec_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG

Phage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo. However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a [~]200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA5-phage) and 50 copies of ScA on p8 protein (ScA50-phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo. A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/432100v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@18edf0org.highwire.dtl.DTLVardef@ad55c6org.highwire.dtl.DTLVardef@1da47d8org.highwire.dtl.DTLVardef@1a2ec32_HPS_FORMAT_FIGEXP M_FIG C_FIG

Current studies of biological tissues require visualizing diverse cell types and molecular interactions, creating a growing need for versatile techniques to simultaneously probe numerous targets. Traditional multiplexed imaging is limited to around five targets at once. Emerging methods utilizing sequential rounds of staining, imaging, and signal removal can probe tens of targets but require specialized hardware, time-consuming workflows, and face some challenges with sample distortion and artifacts. Here we present a new method for highly-multiplexed fluorescence microscopy using semiconducting polymer dots (Pdots) in a single round of staining and imaging. Pdots are small, bright, and photostable fluorescent probes with a wide range of tunable Stokes shifts (20-450 nm). Multiple series of Pdots with varying excitation wavelengths allow for fast (<1 minute) and single-round imaging of up to 21 targets in brain and kidney. This method is based on a simple immunofluorescence workflow, efficient use of spectral space, standard hardware, and straightforward analysis, making it widely applicable for bioimaging laboratories.

Radiopharmaceutical therapy is changing the standard of care in prostate cancer (PCa) and other malignancies. We previously reported high CD46 expression in PCa and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. Our radiolabeled antibody retains binding efficacy and shows a high tumor to background ratio in PCa xenografts. Furthermore, we show that radiolabeled antibody was able to suppress the growth of cell-derived and patient-derived xenografts, including PSMA-positive and deficient models. Nephrotoxicity, not seen at low radioactive doses, is evident at higher radioactivity dose levels, likely due to redistribution of daughter isotope 213Bi. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46 targeted radioligand therapy in PCa.

Surgeries and trauma result in traumatic and iatrogenic nerve damage that can result in a debilitating condition that approximately affects 189 million individuals worldwide. The risk of nerve injury during oncologic surgery is increased due to tumors displacing normal nerve location, blood turbidity, and past surgical procedures, which complicate even an experienced surgeons ability to precisely locate vital nerves. Unfortunately, there is a glaring absence of contrast agents to assist surgeons in safeguarding vital nerves. To address this unmet clinical need, we leveraged the abundant expression of the voltage-gated sodium channel 1.7 (NaV1.7) as an intraoperative marker to access peripheral nerves in vivo, and visualized nerves for surgical guidance using a fluorescently-tagged version of a potent NaV1.7-targeted peptide, Tsp1a, derived from a Peruvian tarantula. We characterized the expression of NaV1.7 in sensory and motor peripheral nerves across mouse, primate, and human specimens and demonstrated universal expression. We synthesized and characterized a total of 10 fluorescently labeled Tsp1a-peptide conjugates to delineate nerves. We tested the ability of these peptide-conjugates to specifically accumulate in mouse nerves with a high signal-to-noise ratio in vivo. Using the best-performing candidate, Tsp1a-IR800, we performed thyroidectomies in non-human primates and demonstrated successful demarcation of the recurrent laryngeal and vagus nerves, which are commonly subjected to irreversible damage. The ability of Tsp1a to enhance nerve contrast during surgery provides opportunities to minimize nerve damage and revolutionize standards of care across various surgical specialties.

In this work we present antibody-metal conjugate as a new subclass of antibody-drug conjugates (ADC) for the chemodynamic therapy of cancer based on the rapid generation of reactive oxygen species (ROS) upon copper reduction. We used conventional therapeutic antibody trastuzumab and DOTA-NHS ester for the design and initial proof-of-concept. Thus, trastuzumab-DOTA-copper conjugate (TDCC) was synthesized. We demonstrate that TDCC retains specific binding to HER2-positive cancer cells with approximately native immunoreactivity and achieves stable copper incorporation with an average drug-to-antibody ratio of up to [~]8. In the presence of physiological reducing agents such as N-acetylcysteine or cysteine, TDCC generates substantial reactive oxygen species (ROS), leading to pronounced cytotoxicity and long-term suppression of clonogenic survival in HER2-positive SK-BR-3 and BT-474 cells. Notably, HER2-negative MDA-MB-231 cells and non-malignant HS5 fibroblasts remain largely unaffected, confirming target-dependent activity. The conjugate remains stable under storage conditions for up to 30 days, and the DOTA linker itself does not interfere with copper-mediated redox chemistry. Our findings identify TDCC as a novel class of targeted oxidative stress inducers that exploit the vulnerability of HER2-positive tumors to copper-mediated cytotoxicity. This strategy not only preserves the specificity of antibody-based delivery but also introduces a distinct mechanism of action capable of bypassing conventional resistance pathways, warranting further preclinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/721915v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@7ed6bdorg.highwire.dtl.DTLVardef@1442b2aorg.highwire.dtl.DTLVardef@6dff28org.highwire.dtl.DTLVardef@18aba16_HPS_FORMAT_FIGEXP M_FIG C_FIG

Proper staining of cell plasma membrane is indispensable for fluorescence imaging. Herein, we present an array of five anionic cyanine-based turn-on plasma membrane probes with emission spanning from green to near infrared. They are analogous of commonly used MemBright probes family, where two zwitterionic anchor groups are replaced with anionic sulfonates with dodecyl chains. The developed probes provide selective wash-free staining of plasma membranes of live cells in vitro, featuring improved brightness and slower internalization inside the cells. In comparison to protein-based (wheat germ agglutinin) membrane markers, new membrane probes provide better staining in poorly assessable cell-cell contacts. A key challenge is to stain cell plasma membranes directly in vivo. During in vivo brain tissue imaging in living mice by two-photon microscopy, the anionic cyanine probes allowed us to visualize in detail the pyramidal neurons with high image quality, clearly resolving neuron soma, dendrites with dendritic spines and axons with axonal boutons. The developed anionic cyanine-based plasma membrane probes constitute an important extension of the toolbox of fluorescent probes for plasma membrane research.