Bioconjugate Chemistry

We have developed a vector platform for delivery of foreign peptides by genetic modification of the temperate lambda ({lambda}) bacteriophage. This delivery platform is capable of displaying peptides or proteins on either terminus of the structural {lambda} head protein D, present in [~]420 copies per phage particle, and {lambda} side tail fiber (Stf), present at 12 copies per phage particle. Proteins and peptides can be easily fused for display through the low-cost and high-efficiency methods of recombineering and {lambda} prophage induction for recombinant phage preparation described here. To improve this vector technology for use in antigen selection and immunotherapy, we introduced several mutations in the bacterial host and resident prophage {lambda} that improve engineering, induction, phage stability, yield, fusion protein accommodation capacity, and longevity in animal systems. We tested the ability of this {lambda} display system to identify useful antigens and generate antibodies in a mouse model. We report its success as a new technology for both applications: the selection and delivery of therapeutic peptides and proteins.

Described here is the synthesis and assembly of a new-generation mRNA-bifunctional lipid nanoparticle (mRNA-BLNP3) for selective delivery of mRNA to antigen-presenting cells (APCs). Compared to mRNA-BLNP1 and BLNP2 (BioRxiv,doi.org/10.1101/2023.12.26.572282), the mRNA-BLNP3, incorporating a glycolipid with the lipid moiety designed to target the CD1d receptor on dendritic cells (DCs) and the sugar head group targeting the mannose-binding receptors on APCs is more selective with stronger target-specific immune responses. It was shown that vaccination in mice with mRNA-BLNP1 elicited enhanced cytokine induction and antibody responses compared to traditional mRNA-LNPs, and the mRNA-BLNP2 vaccine, incorporating a mannose-glycolipid, further improved DC targeting. However, BLNP3, incorporating the glycolipid with an aryl-mannose head group targeting the mannose receptor on APCs and the same lipid moiety targeting the Cd1d receptor on DCs, showed superior lymph node targeting in vivo with reduced liver accumulation, enhanced mRNA expression in DCs and macrophages, and increased DC maturation. Immunization in mice with mRNA-BLNP3 elicited enhanced humoral and cellular immune responses compared to mRNA-BLNP1 and mRNA-BLNP2, with higher antigen-specific IgG titers and granzyme B-producing CD8 T cells, demonstrating that BLNP3 is a promising bifunctional lipid nanoparticle for delivery of mRNA vaccines with improved efficacy and safety.

The contemporary shift toward multispecific antibodies, antibody-drug conjugates (ADCs), and bespoke glycoengineered therapeutics have exposed the limitations of standard genomic engineering tools. This paper presents a novel iterative engineering paradigm utilizing the Leap-In Transposase(R) platform. By leveraging a suite of three mutually orthogonal transposase-transposon systems, we demonstrate the sequential modification of the Chinese Hamster Ovary (CHO) genome to achieve three distinct functional outcomes: (i) First, the creation of a glutamine synthetase (GS)-deficient host (CHO-K1-GS) via targeted knockdown, (ii) Second, the integration of multiple copies of a model therapeutic IgG1 for expression, and (iii) Third, the subsequent knockdown of the fucosylation pathway to modulate the glycan profile of the expressed IgG1. Genetic stability (copy number & sequence) of each integration event was confirmed using Targeted Locus Amplification (TLA) and Next-Generation Sequencing (NGS). Functional stability (expression levels, metabolic phenotype, and glycan phenotypes) was confirmed using standard cell culture and analytical techniques. Crucially, the truly orthogonal nature of the transposase-transposon pairs prevents cross-mobilization and ensures the structural and functional integrity of previously integrated cargo. This study establishes a "What You See Is What You Get" (WYSIWYG) methodology that provides a robust, scalable, and predictable framework for developing next-generation complex biopharmaceutical manufacturing cell lines.

Background/ObjectivesDevelopability assessment is a critical step in advancing antibody-based molecules toward clinical application. This evaluation typically begins during clinical candidate selection and continues throughout all modifications of the molecule during development. It is guided by the target product profile, which includes the intended administration route and regimen, formulation parameters, and process conditions encountered during manufacturing, storage, and delivery. While developability testing is well established for conventional therapeutic antibodies, strategies for assessing single-domain antibodies (sdAbs) and their conjugates remain underexplored. Here we present a strategy to test the developability of sdAbs as a case study for two clinical candidates intended as precursors for the production of diagnostic tracers for clinical imaging. MethodsAssays were developed to evaluate chemical and thermodynamic stability, target binding affinity and capacity, and chelation efficiency ("chelatability"). Accelerated stability studies were conducted for both unconjugated sdAbs and their chelator conjugated forms following incubation at two pH conditions, at multiple time points, and after twelve freeze-thaw cycles to simulate process conditions and long-term storage. Analytical assays were applied stepwise in a hierarchical approach to minimized experimental effort and material consumption. Candidates exhibiting critical developability features were selectively addressed by assays with increasing precision. ResultsA tailored panel of analytical assays optimized for low molecular weight proteins was established and applied to the two clinical candidates, identifying instability hotspots as well as potential mitigation strategies. Successful engineering of a candidate with an initially critical developability profile was achieved. ConclusionThis study demonstrates the implementation of a structured developability assessment strategy for sdAb conjugates. The approach integrates physicochemical and functional stability evaluations, supporting robust candidate selection, formulation development, and method optimization for this class of molecules.

Bifunctional diazirine lipids are valuable tools for mapping protein-lipid interactions and cellular localization by photocrosslinking. Yet, the crosslinking efficiency of these probes has not been systematically evaluated. Here, we use the lipid transfer protein STARD10, which binds phospholipids in a 1:1 stoichiometry within a hydrophobic pocket, to measure the upper limit of the photo-crosslinking efficiency of bifunctional lipid probes. We characterize reaction products using native and denaturing mass spectrometry. Our results show that approximately 5% of photoactivated lipids form covalent protein-lipid crosslinks, while the majority follow intramolecular reaction trajectories, resulting in the formation of products featuring alkene, ketone and hydroxyl moieties. These findings provide essential context for the use of bifunctional probes to uncover the cell biology of lipids and highlight the need for continuous improvement to experimental workflows. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/700185v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@15d1641org.highwire.dtl.DTLVardef@6024e0org.highwire.dtl.DTLVardef@1503dcorg.highwire.dtl.DTLVardef@1b067bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Accurately assessing blood vascular networks is important in all organ systems in health, disease, and healing. However, methods to do so in a holistic fashion in post-mortem specimens have significant limitations. We have previously demonstrated proof of concept for a lead abbelaite nanoparticle and alginate nanocomposite contrast agent which would allow greatly improved vessel imaging using x-ray based imaging modalities like CT and microCT. Specifically the contrast is spectrally enhanced to easily allow segmentation from mineralized tissues and gelation is triggerable permitting better vascular perfusion. Here we expand upon that work by substituting lead tungstate nanoparticles. We found that delivery viscosity and radiopacity are largely unaffected. However, mechanical strength was negatively impacted as abellaite presence was lowered. In sum, these formulations have performed in bulk reasonably enough to warrant advancement to in vivo post-mortem evaluation in small animal models.

Affibodies are remarkably stable three-helix bundle proteins that can be engineered to selectively bind target proteins. When combined with radioactive metals, they serve as imaging agents or cancer therapeutics, depending on the metal used. Traditionally, this involves bifunctional linkers that attach large chelators to the affibody via reactive groups. Here, we present an alternative approach that eliminates the need for such linkers by burying the metal within the core of the affibody, surrounded by its three helices. A simple engineered triple cysteine motif, with one cysteine in each helix, stably binds Bi(III), Pb(II), In(III) and Ga(III), which are commonly used in imaging and radiotherapy. Quantitative metal uptake is instantaneous at room temperature and physiological pH, and all metal-affibody complexes remain fully intact for one week at 4 {degrees}C. All retain their metal cargo when challenged with cellular concentrations of glutathione, while only the bismuth-affibody complex withstands a challenge with 100 equivalents of strong chelators, even over two weeks. We demonstrate selective uptake and retention of 213Bi, a promising isotope for targeted alpha therapy.

The rapid engineering of high-affinity binding proteins, such as nanobodies and single-domain antibodies (sdAbs), is increasingly driven by cell-free, machine-learning-guided optimization. However, high-throughput, quantitative characterization of binding affinity remains a major bottleneck, particularly for proteins expressed in cell-free systems without purification. Here, we present High-Throughput FRET Affinity Screening Technique (HTFAST) for rapid affinity characterization of binders expressed directly in crude E. coli cell-free protein synthesis reactions. HTFAST leverages Forster resonance energy transfer (FRET) between fluorescent-protein-fused binders and dye-labeled antigens to enable real-time, quantitative measurement of equilibrium dissociation constants. We systematically optimized fluorophore pairs used and labeling parameters using the SpyTag003-SpyCatcher003 model system. Using donor-quenching and acceptor-emission FRET analyses, HTFAST reliably quantified nanomolar binding affinities in crude lysates for SpyTag003-SpyCatcher003 model system. We validated the platform for nanobodies by characterizing a CD4-binding nanobody, Nb457, and benchmarking multiple SARS-CoV-2 receptor-binding domain sdAbs, demonstrating HTFASTs ability to rank binding strengths across a range of affinities. Finally, we demonstrate that both binding partners can be expressed directly in CFPS, further streamlining screening workflows. Overall, HTFAST provides a scalable, quantitative, and cell-free-compatible approach for high-throughput affinity screening, well suited for DBTL campaigns aimed at accelerating the development of next-generation binding proteins.

Fibroblast activation protein (FAP) is an attractive target for the development of cancer theranostics due to its selective expression on cancer-associated fibroblasts (CAFs). While a number of small-molecule FAP inhibitors (FAPIs) have been developed, few biologics have been investigated as FAP targeting vectors. Camelid-derived single-domain antibodies, or variable-heavy-heavy domains (VHHs), offer a compelling alternative, combining high affinity with versatile engineering options. In this study, we first identified a novel anti-FAP VHH, F7, from an affinity-matured camelid phage display library. To investigate how valency and molecular weight affected target engagement and in vivo properties, F7 was engineered into three formats: a monomer (F7), a tethered dimer (F7D), and an Fc-fusion protein (F7-Fc). All three were specific for FAP with the two bivalent constructs demonstrating picomolar affinity. Positron emission tomography imaging in FAP-positive xenograft models revealed distinct pharmacokinetic profiles across constructs with notable differences in tumor uptake and clearance. F7 had rapid uptake and clearance resulting in significantly higher tumor uptake than FAPI-46. Low molecular weight bivalent F7D demonstrated similar kinetics but was retained by the tumor resulting in a high tumor-to-blood ratio with secondary uptake limited to clearance organs. The largest construct, F7-Fc, resulted in the highest tumor uptake and allowed for longitudinal imaging. Absorbed dose calculations confirmed that tumors received significantly higher radiation doses compared to normal tissues. These findings demonstrate that tuning VHH scaffold size and valency can improve biodistribution and retention, establishing F7-based constructs as promising targeting vectors for FAP.

Glioblastoma (GBM) is an aggressive, high-grade glioma with a near-universally fatal prognosis. Therapeutic failure is often attributed to the highly selective blood brain barrier (BBB), the diffuse infiltrative nature of the tumor, and the marked intratumoral heterogeneity of GBM. Although antibody drug conjugates ADCs have shown promise for high grade gliomas such as GBM, efficacy is limited by ADC size. Aptamers--short, synthetic, single-stranded DNA or RNA molecules--can be [~]6-fold lower in molecular weight than IgG antibodies and have the potential to cross the intact BBB. Unlike other nucleic acid-based therapies, aptamer function arises from three-dimensional shape rather than genetic coding. Here we aim to replace the targeting component of the ADC paradigm with a DNA aptamer, thus creating an aptamer-drug conjugate (ApDC). We employed in vivo SELEX using an orthotopic patient-derived xenograft (PDX) GBM mouse model and a vast ([~]100 trillion 80-mer sequences) ApDC library. We report the results from this first in vivo ApDC selection of its kind. We characterize target tissue binding ex vivo, cell association, biodistribution, and pharmacokinetics from this selection. This study exemplifies an unbiased approach to a problem that rational design has yet to overcome, offering a new direction for GBM therapeutic development.

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors including small cell lung cancer (SCLC) and represents a validated target for peptide receptor radionuclide therapy. The SSTR2 agonist [177Lu]Lu-DOTA-TATE is clinically approved, however, treatment resistance and relapse occur. The SSTR2 antagonist SSO110 (DOTA-JR11, OPS201) demonstrates higher tumor uptake and longer retention than DOTA-TATE both pre-clinically and clinically. We performed a systemic head-to-head comparison of SSO110 labeled with various radionuclides of distinct emission characteristics to identify the optimal radionuclide for SSO110 and to compare antagonist with agonist performance. MethodsSSO110 was radiolabeled with 177Lu, 161Tb, 212Pb, and 225Ac. Biodistribution was assessed in AR42J and NCI-H69 xenograft models. Therapeutic efficacy of single and fractionated [212Pb]Pb-SSO110 was compared with [177Lu]Lu-SSO110 in NCI-H69 tumors. Single-dose efficacy of 225Ac-, 161Tb-, and 177Lu-labeled SSO110 was evaluated in both models. [{superscript 2}{superscript 2}Ac]Ac-DOTA-TATE served as agonist comparator. Tumor growth, survival, safety parameters, and tumor absorbed doses were analyzed. ResultsAll SSO110 radioconjugates demonstrated comparable biodistribution with high tumor uptake and favorable tumor-to-kidney ratios. In NCI-H69 tumors, [212Pb]Pb-SSO110 induced dose-dependent tumor growth delay but did not improve anti-tumor efficacy compared with [177Lu]u-SSO110 under single or fractionated regimens. [161Tb]Tb-SSO110 showed efficacy comparable to [177Lu]Lu-SSO110 in NCI-H69 model and significantly improved tumor growth delay in high-SSTR2-expressing AR42J tumors. Across both models, [225Ac]Ac-SSO110 demonstrated the highest therapeutic potency, inducing durable tumor regression and 100% survival at clinically relevant activities. [225Ac]Ac-SSO110 also outperformed the agonist comparator [225Ac]Ac-DOTA-TATE. Dosimetry analysis revealed a 63-fold higher tumor absorbed dose per injected administered activity for [225Ac]Ac-SSO110 compared with [212Pb]Pb-SSO110. All treatments were well tolerated without significant renal or hepatic toxicity. ConclusionTherapeutic efficacy of SSTR2-targeted peptide receptor radionuclide therapy appears to benefit from alignment between radionuclide physical half-life and ligand tumor residence time. Among the radionuclides evaluated, [225Ac]Ac-SSO110 demonstrated the most pronounced and durable anti-tumor efficacy, outperforming [161Tb]Tb-SSO110, [177Lu]Lu-SSO110, and the short-lived -emitter [212Pb]Pb-SSO110. These findings support clinical investigation of [225Ac]Ac-SSO110 in SSTR2-positive malignancies.

Genetically encoded calcium ion (Ca2+) indicators (GECIs) enable visualization of Ca2+ dynamics in living systems but often suffer from limited photostability during prolonged imaging. The recent discovery of StayGold, a green fluorescent protein (FP) with exceptional brightness and photostability, opened the possibility of addressing this longstanding challenge. Here, we sought to establish whether a monomeric variant of StayGold (mStayGold) could be converted into a single FP-based GECI. Through extensive protein engineering, we generated a functional mStayGold-based GECI, HiCaRI (Highly intensiometric Ca2+ Responsive Indicator) by fusing Calmodulin (CaM) and the ckkap binding peptide from K-GECO1 into mStayGold(J). HiCaRI exhibits a large Ca2+-dependent inverse fluorescence response ({Delta}F/Fmin = -15) while retaining high brightness and improved photostability relative to previously reported GFP-based GECIs. Although the current variant represents a first-generation prototype with shortcomings in terms of Ca2+ affinity and photostability (relative to StayGold and mStayGold(J)), this work demonstrates the feasibility of constructing single FP-based GECIs from a highly photostable fluorescent protein.

Both for diagnostic purposes and regenerative medicine, it is essential to develop advanced imaging platforms capable of tracking the biodistribution of small extracellular vesicles (sEVs), as current methods are limited by inadequate resolution and sensitivity. In this study, we introduce a novel labeling strategy utilizing the radioisotope zirconium-89 (89Zr), which boasts a half-life of 78.4 h and is cost-effective to produce. To achieve this, we designed a new chelator tailored for 89Zr4+ that offers enhanced stability compared to the conventional deferoxamine (DFO). This chelator forms a robust complex with 89Zr4+ at room temperature, suitable for sEV labeling for PET imaging applications. The radiolabeling process involved a two-step procedure: first, conjugation of the chelator to the sEVs, and second, radiolabeling with 89Zr4+. The resulting sEV-L1-Zr demonstrated a radiochemical yield of approximately 60% and maintained around 80% stability in plasma over seven days. Importantly, our modifications did not alter the morphology, surface protein composition, internal RNA content, or bioactivity of the sEVs. We successfully visualized sEVs at very low doses in the mouse heart following intravenous injection of sEV-L1-Zr. Additionally, ex vivo experiments using a Langendorff rat heart perfusion model confirmed targeted accumulation of the vesicles in cardiomyocytes as compared to other cells in the heart compartment. This approach provides a promising platform for sensitive and stable in vivo tracking of sEVs, advancing their application in both diagnostic imaging and regenerative therapies.

mRNA-lipid nanoparticles (LNP) have proven their potential as a rapidly adaptable vaccine platform and promise to revolutionize numerous therapeutic areas. A major hurdle towards the widespread adoption of mRNA-LNP vaccines and therapeutics is their limited liquid shelf-life compared to more established modalities currently necessitating an ultralow temperature cold-chain to enable their distribution and storage. While ongoing efforts aim to improve liquid stability through chemical modification of mRNA and lipid components, complementary strategies that are broadly applicable across chemistries may further accelerate translation. Here, we present an approach to improve the liquid shelf-life of mRNA-LNPs that does not rely on modifications to the mRNA or LNP chemistry. In particular, we show that bleb formation induced by high ionic strength acidic citrate buffers during LNP formation reduces mRNA degradation and retains in vitro activity during extended liquid storage. We observed an increase in the in vitro activity storage half-life from 2.8 to 18.9 days at 25{degrees}C when prepared using high ionic strength buffers translating into a [~]7-fold improvement in the liquid shelf-life of MC3-LNPs. This enhanced stability of LNPs with large amount of bleb formation was mainly attributed to reduced rates of lipid-mRNA adduct formation and mRNA fragmentation. Furthermore, the acidic buffer dependent stabilization was observed across different ionizable lipids with the extent dependent on the ionizable lipid head group. We envision that the induction of bleb formation via selection of appropriate acidic mixing buffers may represent a universal approach to enhance mRNA-LNPs stability and enable extended long-term refrigerated storage.

Virus-like particles (VLPs) represent a promising next-generation drug delivery platform. However, conventional VLPs rely on multiple viral components for effective cargo encapsulation and delivery, raising safety concerns. Here, we present a novel strategy to engineer immature VLPs using a self-cleaving intein system. We employed viral Gag proteins as sorting domains, linking cargo proteins to Gag through inteins, thereby eliminating the need for the conventional protease cleavage typically mediated by the gag-pol protein. During VLP biogenesis, intein-mediated cleavage released cargo proteins into the lumen, enabling efficient intracellular delivery when VLP surfaces are pseudotyped with VSV-G. Optimal candidates for delivering Cre recombinase and gene editing tools (Cas9, Cas12a and base editors) were identified by screening various Gag proteins. Notably, these VLPs achieved robust gene editing in primary cells, including naive and activated T cells, as well as hematopoietic stem and progenitor cells (HSPCs). A single local intracerebroventricular (ICV) infusion of optimized particles induced up to 60% tdTomato expression in the brain regions of reporter mice, while intravenous injection resulted in significant recombination (up to 70%) of a variety of cell types across organs. Collectively, we developed a simplified, efficient VLP platform for intracellular cargo delivery with broad therapeutic potential for gene editing and treatment of human diseases.

Peritoneal micrometastases (micromets) remain a major barrier to durable cytoreduction in ovarian and other intra-abdominal cancers, because lesions can be difficult to visualize and are often resistant to systemic therapy. Liposomal doxorubicin (Dox) improves pharmacokinetics but can be limited by slow intratumoral release. Porphyrin-phospholipid (PoP) liposomes enable near-infrared light-triggered release of Dox (chemophototherapy (CPT)), creating an opportunity for intraoperative, fluorescence-guided treatment planning and monitoring. Here, we evaluate a laparoscopic fluorescence imaging platform for quantifying light-triggered drug delivery in 2D monolayers and 3D spheroid cluster models. Dox fluorescence increased linearly with administered LC-Dox-PoP concentration in both SCC2095sc and SKOV-3 cultures (R2 = 0.97-0.98 in 2D; R2 = 0.98 in spheroid clusters over 1-9 {micro}g/mL). Laparoscope-derived fluorescence measurements agreed with standard well-plate reader measurements (R2 = 0.89-0.96). Porphyrin fluorescence provided stronger, complementary contrast for localizing spheroid constructs and decreased after activation light exposure, consistent with photobleaching during triggered release. Together, these results support a quantitative imaging framework for fluorescence-guided monitoring of light-triggered liposomal drug release, with potential to inform individualized CPT dosimetry for peritoneal micrometastases. These findings in SCC2095sc (oral squamous cell carcinoma) additionally suggest relevance of fluorescence-guided CPT for head and neck/oral cancer, where localized post-resection adjuvant treatment may improve control of residual disease.

CLIP-tag is a self-labeling protein tag used for the specific fluorescence labeling of proteins. However, its low labeling speed and the poor cell permeability of its substrates result in low labeling efficiencies in live-cell applications. Here, we introduce a substrate optimized for live-cell applications, as well as an engineered CLIP-tag variant, CLIP-tag2, which reacts with the new substrate almost 1000-fold faster than the original CLIP-tag-substrate pair. CLIP-tag2 fusion proteins can be specifically and efficiently fluorescently labeled in cells within minutes at nanomolar substrate concentrations, and can be multiplexed with other self-labeling tags such as SNAP-tag2 and HaloTag7. These advances establish CLIP-tag2 as a powerful tagging platform for high-performance live-cell bioimaging.

Poly(ADP-ribose) polymerase 1 (PARP1) is a central mediator of DNA damage repair and an established therapeutic target in homologous recombination-deficient cancers. Radiolabelled PARP inhibitors provide a strategy to deliver cytotoxic radiation directly to tumour DNA by exploiting PARP overexpression and trapping at sites of DNA damage. Here, we describe the design, radiosynthesis, and in vitro evaluation of [123I]Italia, a talazoparib-derived Auger electron-emitting agent for PARP-targeted radionuclide therapy. Stereochemically pure [123I]Italia, (8S,9R)-5-fluoro-8-(4-(iodo-123I)phenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one was synthesised in one step via copper-mediated iodo-deboronation, achieving activity yields >80% and molar activities >6.2 {+/-} 3.1 GBq/{micro}mol (n=8). UPLC analysis confirmed radiochemical purity >97%. Italia exhibited potent PARP1 inhibition (IC50 0.48 nM) and in silico predicted binding affinity comparable to talazoparib. In a panel of PARP-expressing cancer cell lines, [123I]Italia demonstrated highest uptake at 60 min, PARP-selective uptake, predominant nuclear localisation (up to 60% of added activity) and chromatin association consistent with PARP trapping (up to 15% of total activity recorded). Uptake was reduced more than 50-fold by addition of an excess of any PARP inhibitor (e.g. olaparib, talazoparib, and rucaparib) and in PARP1 knockout cells, confirming target specificity. Clonogenic assays showed a marked, added activity-dependent reduction in survival of PARP-expressing cells following a brief one-hour exposure, whereas PARP1-deficient cells were resistant. Collectively, these findings identify [123I]Italia as a promising PARP-targeted Auger electron-emitting theranostic candidate that warrants further in vivo evaluation.

Here, we report, for the first time, delivery of mRNA and/or protein payloads into primary adult human hematopoietic stem cells (HSCs) using a bacteriophage-derived nanoparticle vector. We have been developing a new category of bacteriophage T4-engineered nanoparticles, termed "artificial viral vectors" (AVVs), for delivering therapeutic nucleic acid and protein complexes into human cells. Using a defined in vitro assembly-line platform, we decorated the capsid surface with mRNA and protein complexes through two outer capsid proteins, Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein). First, we displayed a Hoc-protein G fusion protein to which HSC-targeted monoclonal antibodies (mAbs) are attached. Then we decorated the nanoparticle with the Soc-fused HIV-TAT molecule to create a positively charged capsid surface with cell penetrating function. Reporter mRNA molecules are then displayed on the capsid surface and the nanoparticle is coated with lipids. Such T4-AVVs transduced HSCs and delivered GFP and Luciferase reporter mRNAs at levels as high as 20% efficiency and exhibited targeted Ab-dependent phenotypes. Furthermore, we demonstrate simultaneous delivery of both protein and mRNA payloads, by decorating the capsid with mRNA and [~]507 kDa tetrameric {beta}-galactosidase. The AVV-transduced HSCs maintain viability, and once optimized, the T4-AVV platform will provide enormous versatility to target various types of human cells and deliver next generation therapies for cancer and genetic diseases.

Fluorescent proteins (FPs) are essential tools for biological imaging but are limited by photobleaching, a light-induced loss of fluorescence intensity that reduces spatial and temporal resolution. Despite extensive use, the molecular mechanisms underlying FP photobleaching remain poorly understood due to the diversity of FPs and the complexity of their photochemistry. Existing approaches either monitor fluorescence decay in live cells, reflecting imaging conditions but lacking molecular detail, or rely on in vitro spectroscopy of purified proteins, providing mechanistic insight but often limited to individual FPs. We introduce a quantitative workflow bridging these approaches by combining live-cell measurements with in vitro spectroscopy. In vitro measurements are performed on a dedicated setup that simultaneously monitors absorption, emission, and fluorescence decay during photobleaching. Applied to six FPs spanning different chromophores, emission ranges and sequences, this approach reveals that photobleaching strongly depends on FP. It involves multiple chemical pathways, including oxidation, dimerization, and backbone cleavage. Spectroscopic analysis uncovers a heterogeneous ensemble of photoproducts with distinct photophysical properties that can remain optically active during irradiation, including shortened fluorescence lifetimes or altered absorption spectra. These findings demonstrate that FP photobleaching cannot be described as a simple ON-OFF process but involves complex transformations affecting both fluorescence intensity and lifetime. Such transformations can introduce significant biases in quantitative imaging, particularly in advanced techniques such as FLIM and FRET. Finally, we introduce quantitative indicators enabling robust comparison of FP photostability across experimental conditions. This framework provides a comprehensive approach for understanding and quantifying photobleaching and its implications for fluorescence imaging.