Notebook

Introduction

Our project started from April to September, focusing on the development and assessment of bionic fluorescent proteins labeled with near-infrared type-II (NIR-II) dyes for advanced biomedical imaging. The research was meticulously planned and executed over several months, beginning with foundational laboratory training and culminating in the establishment of mouse models for in vivo imaging.

April

    Week 1: Laboratory Access Training and Assessment

This week, we participated in safety training at the Translational Medicine Research Institute of Jilin University, covering access qualifications for cell rooms, animal facilities, and public instrument platforms. The training encompassed laboratory safety regulations, animal experimentation procedures, rules for entering and exiting the animal facilities, and approaches to handling major safety issues.

    Weeks 2 & 3: Basic Experimental Skills Training

During this period, we gained familiarity with the laboratory's equipment and fundamental experimental operations, including cell culture, bacterial culture, and the utilization of instruments such as shakers, incubators, microplate readers, and UV-Vis spectrophotometers. We had our first attempt at Escherichia coli cultivation.

    Week 4: Experimental Design and Discussion in Detail

Collaborating with our supervising instructor, we deliberated on the experimental plan and its specifics, clarifying the primary objectives and anticipated outcomes. We formulated a detailed timeline, outlining the time nodes and critical tasks for each experimental phase.

May: constructing bionic fluorescent proteins

    Weeks 1 & 2: Screening of Protein-Labeling NIR-II Dyes

Given the current lack of NIR-II dyes that can directly and covalently label proteins, we embarked on a screening process involving various common NIR-II dyes such as IR-26, IR-1061, Et-1080, and CO-1080. These dyes were co-incubated with potential protein carriers in vitro. By adjusting factors like reaction time, temperature, and feed ratio, we successfully identified that CO-1080 could bind relatively quickly with human serum albumin under mild conditions.

    Week 3: Exploring Structures and Binding Capabilities

To gain deeper insights into why different dye molecules exhibit varying binding efficiencies with proteins, we attempted to uncover the relationship between molecular structures and their affinity towards proteins using molecular docking methods.

    Week 4: Quantifying Binding Capabilities

To accurately assess the construction efficiency of biomimetic fluorescent proteins, we employed protein electrophoresis and high-resolution mass spectrometry to isolate and determine the precise proportion of the protein-bound NIR-II luminescent core (CO-1080) within the mixture before and after binding. The results indicated that although the protein could be almost completely labeled by the dye under heated conditions, further optimization was necessary to reduce the reaction temperature and time, which were higher and longer than those encountered in living organisms.

June: Identification of Binding Sites

    Weeks 1 & 2: Confirmation of Dye-Protein Binding Sites

Based on previous reports, albumin is known to have three structural domains. We obtained recombinant proteins of these three domains and incubated them with the dye in vitro, observing their brightness under an NIR-II camera. The results revealed that one particular domain significantly enhanced the dye's fluorescence. Additionally, gel electrophoresis indicated that this domain could fully bind with the dye to form a stable complex.

    Weeks 3 & 4: Proteomic Identification of Specific Binding Sites

Proteomic analysis was performed on the resulting biomimetic fluorescent proteins using Nano LC-MS/MS to identify the specific amino acid residue sites where the dye was bound.

July: Preparation of Mutant Proteins

    Week 1: Design of Mutant Protein Sequences

scores, consistent with our prediction that the reaction between the dye and protein occurs at the thiol groups of cysteines. However, we also noted that these sites primarily form disulfide bonds within the protein structure. After discussing with our supervisor, we planned to mutate these amino acid sites to achieve more efficient protein-dye binding.

    Weeks 2 & 3: Confirmation of Experimental Feasibility and Preparation

This week, we purchased the necessary reagents and consumables, learned the relevant protein expression techniques, and initiated experiments for protein expression.

    Week 4: Continued Optimization of Mutant Protein Preparation and Successful Expression of Small Quantities

We successfully obtained small quantities of mutant proteins and co-incubated them with the dye. The addition of mutant proteins accelerated the reaction rate between the dye and protein, enabling the preparation of biomimetic fluorescent proteins at 37°C.

August: Assessment of Dye Biocompatibility and In Vivo Imaging Capabilities

    Weeks 1 & 2: Biocompatibility Assessment

Materials intended for direct use in biological systems must undergo thorough biocompatibility assessments. This week, we cultured several types of common cells, including normal tissue cells and cancer cells. MTT assays were performed to verify whether the biomimetic fluorescent proteins had acceptable toxicity levels towards these cells. Subsequently, the proteins were injected into mice, and their acute and chronic toxicity within the animals was evaluated through blood tests (routine, liver function, kidney function), and histopathological examinations of major organs.

    Week 3: Systematic Evaluation of Metabolic Capabilities Considering Potential Clinical Applications

We assessed the metabolic profiles of different dyes, determining the blood half-lives of the biomimetic fluorescent proteins and the organ distribution of their fluorescent signals.

    Weeks 4: In Vivo Imaging Capabilities Assessment

After administering the biomimetic fluorescent proteins through footpad or tail vein injection, mice were immediately imaged using an NIR-II camera for visualization of blood vessels and lymphatic vessels. Imaging capabilities were evaluated by quantifying light-up times and signal-to-noise ratios.

September: Mouse Model Establishment and Imaging

    Week 1: Mouse Model Construction

We experimented with varying laser irradiation durations to determine the optimal level of barrier disruption for achieving the best imaging outcomes and detection thresholds.

    Weeks 2 & 3: In Vivo Imaging

Following model establishment, mice were injected with different biomimetic fluorescent proteins and unmodified free dyes. Under NIR-II imaging, the fluorescent signals in the modeled regions were continuously observed and quantified. After the experiments, mice were euthanized, and tissues were harvested for quantitative analysis of fluorescent leakage in both damaged and normal tissues.

    Week 4: Data Compilation and Analysis

All data were compiled, and their relevance and reliability were verified.

Weekly Notebook

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