Results
We present the results according to the project's design logic, proceeding from genetic construction to functional validation and, finally, to product integration. This section is structured into three main parts: 1) the successful construction of engineered yeast strains, 2) the functional efficacy of the multi-layered therapy system, and 3) the performance of the smart encapsulation and delivery system.
1. Successful Construction of Engineered Yeast Strains
The synthesized plasmids, hosted in E. coli, were extracted using a commercial kit. As the vendor provided a quality report, re-sequencing was omitted. We then transformed the four target plasmids—carrying the Pexiganan surface display system, VEGF, IL-4, and IL-10 genes—into the Yarrowia lipolytica Po1h strain (Genotype: Ura-, ΔAEP, ΔAXP, Suc+). As the strain is auxotrophic for uracil, the plasmids carried the Ura3 gene, allowing positive transformants to be selected on YNB medium. Several viable single colonies were successfully obtained. Selected single colonies were cultured in YPD medium for expansion, followed by genomic DNA extraction from the transformants. PCR amplification was performed for each target gene, and the products were analyzed by agarose gel electrophoresis to confirm the size of the amplified genes.
Confirmation of successful genomic integration for all four target genes was obtained by agarose gel electrophoresis of PCR products from the Yarrowia lipolytica Po1h transformants. Img.1 shows distinct bands of the expected sizes for Pexiganan (483 bp), VEGF (673 bp), IL-4 (481 bp), and IL-10 (541 bp), validating the successful integration and presence of the target genes in our engineered strains.
.
Img.1 Construction validation of the engineered Yarrowia lipolytica strains for diabetic wound therapy. Cloning verification of the key therapeutic genes in the recombinant Yarrowia lipolytica strains. Specific bands amplified by colony PCR from positive clones, with the electrophoresis results confirming the successful construction and transformation of the expression vectors into the yeast cells. (a) Gene encoding the antimicrobial peptide Pexiganan (483 bp) for surface display. (b) Gene encoding vascular endothelial growth factor (VEGF, 673 bp) under the control of a heat-inducible promoter. (c) Gene encoding interleukin-4 (IL-4, 481 bp) under the control of a glucose-inducible promoter. (d) Gene encoding interleukin-10 (IL-10, 541 bp). The leftmost lane in each panel is the DNA molecular weight marker. It is important to note that while the IL-10 gene was successfully cloned, the corresponding engineered strain failed to express the protein at a detectable level and was thus excluded from subsequent functional experiments.
2. Therapy System
2.1 Functional Surface Display of Antimicrobial Peptide Pexiganan
Since Pexiganan is designed to be firmly displayed on the cell wall and not secreted into the supernatant, we could not isolate it from the culture medium. The standard method for verifying surface display via plasmid is to fuse the target protein with GFP and observe fluorescence under a microscope. Due to time constraints, we opted to demonstrate Pexiganan's functionality as an indicator of successful surface display.
We evaluated the antibacterial capability using the Oxford cup method, which revealed potent antibacterial activity of the engineered yeast. As illustrated in Img.2, the culture of Pexiganan-displaying yeast generated a clear inhibition zone with a diameter of approximately 1.6 cm against both E. coli and S. aureus, confirming the successful surface display and functionality of the antimicrobial peptide.
Img.2 Validation of the antibacterial activity of engineered yeast surface-displaying Pexiganan. Antibacterial assessment of the engineered yeast using the Oxford cup method on a single agar plate. (a) Zone of inhibition observed after adding 100 μL of Yarrowia lipolytica culture surface-displaying the antimicrobial peptide Pexiganan. The clear zone demonstrates the effective bactericidal activity of the engineered yeast. (b) Control area without an Oxford cup, showing the normal growth of Escherichia coli and Staphylococcus aureus in the absence of any intervention.
2.2 Secretion of Bioactive Anti-inflammatory Factor IL-4
2.2.1 Bioactivity of IL-4
Given the structural complexity of interleukins, we prepared two factors as a precaution. Since our goal was to induce M2 macrophage polarization to alleviate inflammation, and both IL-4 and IL-10 can promote this polarization, successful expression of either one would suffice. Positive transformants verified by PCR were induced in PPB medium for 3 days to express the target proteins from the IL-4 plasmids, respectively. The expression products were precipitated using ice-cold acetone and initially detected by PAGE.
We set up three experimental groups: a group treated with IL-4 secreted by yeast, a positive control group with commercial IL-4, and a group treated with LPS. Equal volumes of reagents and media were added and co-cultured with RAW264.7 cells. The M1/M2 ratio was detected by flow cytometry after one day. The results demonstrated that the IL-4 secreted by the engineered yeast was biologically active (Img.3).
Img.3 Effect of IL-4 secreted by engineered yeast on macrophage polarization. Flow cytometric analysis of the M1/M2 phenotype ratio in RAW264.7 macrophages under different treatments.
Compared to the pro-inflammatory state induced by LPS (M1/M2 ratio > 3), both the addition of 20 ng/mL commercial IL-4 and 100 μg/mL crude protein (containing IL-4) secreted by the engineered yeast significantly promoted macrophage polarization towards the anti-inflammatory M2 phenotype, reducing the M1/M2 ratio to below 1, confirming the IL-4 in the crude protein holds full bioactivity.
2.2.2 Selection of Glucose-inducible Promoter
To identify a promoter exhibiting strong responsiveness to glucose, three glycolysis-related genes were selected and analyzed by qRT-PCR. We first assessed the integrity of the extracted RNA by electrophoresis (Img.4), which revealed sharp ribosomal RNA bands. The results identified the PGK promoter as the most responsive, exhibiting the strongest induction of downstream gene expression under high glucose conditions (Img.5).
Img.4 Yeast RNA samples for promoter screening.
Agarose gel electrophoresis of total RNA from yeast cultured under high-glucose and glycerol-replacement conditions. The RNA samples from both groups exhibited a characteristic electrophoretic profile dominated by distinct 18S and 5S rRNA bands. These samples were subsequently used as templates in qPCR analyses, which successfully identified a glucose-sensitive promoter, confirming their suitability for this transcript-level analysis.
Img.5 Analysis of glycolysis-related gene expression in response to high glucose.
The relative expression levels of three glycolysis-related genes (PGK, TDH, HXK) in Yarrowia lipolytica cultured under high-glucose versus glycerol (control) conditions were analyzed by qRT-PCR. Data were normalized to the expression levels under glycerol culture (set as 1).
As shown, the PGK gene exhibited the most significant upregulation in response to high glucose, with its expression increasing more than twofold. In contrast, the expression of TDH and HXK showed markedly lower induction (both less than 1.4-fold). This result clearly demonstrates the high glucose sensitivity of the PGK promoter, leading to its selection for driving the expression of the anti-inflammatory cytokine IL-4.
2.2.3 Validation of promoter PGK
To validate the functionality of the PGK promoter, we constructed a pPGK-GFP expression cassette. As shown in Img.6, the engineered yeast harboring this plasmid exhibited green fluorescence under microscopy, with significantly greater intensity than the wild-type control, demonstrating that the PGK promoter effectively drives recombinant gene expression.
Img.6 Functional validation of glucose-induced GFP expression driven by the PGK promoter.
Fluorescence microscopy analysis of GFP expression in engineered yeast harboring the pPGK-GFP plasmid under high-glucose conditions. The top left panel shows a bright-field image of wild-type yeast, and the top right panel shows the corresponding fluorescence image, showing only minimal background signal. The bottom left panel shows a bright-field image of engineered transformants, and the bottom right panel shows their corresponding fluorescence image. Upon excitation at 492 nm, a strong green fluorescent signal is observed in the transformants, which is absent in the wild-type control. This result visually confirms that the PGK promoter is effectively activated by glucose and capable of driving robust heterologous gene expression.
2.3 Secretion of Pro-angiogenic VEGF with Demonstrated Bioactivity
2.3.1 Bioactivity of VEGF
To directly assess its bioactivity, we measured the ability of yeast-secreted VEGF to promote cell proliferation. After synchronizing the cell cycle using CCK-8, we set up two groups: one treated with VEGF secreted by yeast and a blank control group. Cell proliferation was then quantified by measuring the OD450 using a microplate reader.
Img.7 Cell proliferation-promoting effect of yeast-secreted products.
The impact of crude protein secreted by engineered yeast on cell proliferation was assessed using a CCK-8 assay. After 48 hours of culture, cell viability was quantified by measuring the OD450. The group supplemented with the crude protein (containing yeast-secreted VEGF) exhibited a significantly higher OD450 value than the blank control group (p < 0.01), indicating enhanced HUVEC proliferation. This quantitative result confirms that our engineered yeast secretes VEGF with significant mitogenic activity.
2.3.2 Selection of Heat-inducible Promoter
Similar to the glucose-sensitive promoter, to identify a promoter with a strong response to heat stimulation, we selected three heat shock protein-related genes and analyzed their expression levels using qRT-PCR. The results showed that the HSP90 promoter was the most effective, with the highest upregulation of its downstream gene expression (Img.8).
Img.8 Expression response of heat shock genes to different temperatures. The relative expression levels of HSP40, HSP70, and HSP90 genes in Yarrowia lipolytica at 37°C, 39°C, and 40°C were analyzed by qRT-PCR.
demonstrated the most potent induction at all tested temperatures, with a dramatically stronger response compared to HSP40 and HSP70. Notably, at 39°C, HSP90 expression exhibited an exceptional, orders-of-magnitude upsurge, exceeding 700-fold relative to the baseline. In stark contrast, the induction of both HSP40 and HSP70 was minimal (less than 5-fold). Consequently, the HSP90 promoter was selected to construct the infrared light-triggered pro-angiogenic genetic circuit.
2.3.3 Validation of promoter HSP90
A functional analysis of the HSP90 promoter was conducted by constructing an pHSP90-GFP expression cassette. Following thermal induction at 39°C, transformants displayed a distinct and significantly elevated fluorescent signal relative to the negligible background of the wild-type control, conclusively establishing the efficacy of the HSP90 promoter under heat-shock conditions.
Img.9 Functional validation of heat-induced GFP expression driven by the HSP90 promoter.
Fluorescence microscopy analysis of GFP expression in engineered yeast harboring the pHSP90-GFP plasmid following heat shock at 39°C. The top left panel shows a bright-field image of wild-type yeast, and the top right panel shows the corresponding fluorescence image, revealing only faint autofluorescence. The bottom left panel shows a bright-field image of engineered transformants, and the bottom right panel shows their corresponding fluorescence image. Upon excitation at 492 nm, a strong green fluorescent signal is observed in the heat-shocked transformants, which is absent in the wild-type control. This result visually confirms that the HSP90 promoter is effectively activated by thermal stress, establishing it as the core regulatory component for the programmed control of pro-angiogenic factor (VEGF) expression.
3. Encapsulation and Delivery System
3.1 Characterization of the Thermosensitive Properties of L-HBC
The thermosensitive properties of L-HBC were characterized using a rheometer. The temperature was gradually increased from 4°C to 50°C. The gelation temperature, defined by the crossover point of the loss (G'') and storage (G') moduli, was determined to be 20.3°C. Given the human body temperature of 37°C, this formulation can rapidly transition from a liquid to a gel state at the wound site. However, during the mixing process with yeast, it does not gel immediately, making it convenient for application.
Img.10 Temperature-dependent rheological behavior of the L-HBC hydrogel. Dynamic rheological analysis of 3% (w/v) L-HBC hydrogel during a temperature ramp from 4°C to 50°C.
The graph shows the evolution of the storage modulus (G', representing elastic solid-like behavior) and loss modulus (G'', representing viscous liquid-like behavior) as a function of temperature. The crossover point of the two curves (i.e., the gelation point) is identified at 20.3°C. Below this temperature, G'' > G', indicating a liquid state; above it, G' > G'', confirming the formation of a solid gel. This result verifies the excellent thermos-sensitivity of L-HBC. Its gelation point, being significantly below body temperature (37°C), ensures a rapid sol-gel transition upon injection into wounds, allowing it to perfectly conform to irregular wound beds.
3.2 Encapsulation Efficiency of L-HBC with Pexiganan-Expressing Transformants
Encapsulation efficiency was evaluated by monitoring yeast leakage into PBS from the L-HBC hydrogel over 72 hours (Img.11). The system demonstrated excellent containment within the first 24 hours, with zero leakage observed. By 48 hours, minimal leakage was detected (4 colonies), and the number of leaked colonies increased to 26 by 72 hours. This time-dependent leakage profile provides an evidence-based guideline for dressing replacement, confirming a safe therapeutic window of at least 24-48 hours.
Img.11 Time-course evaluation of engineered yeast encapsulation and leakage within the L-HBC hydrogel; (a) 24-hour leakage; (b) 48-hour leakage; (c) 72-hour leakage
These results demonstrate that our encapsulation strategy achieves complete containment within the first 24 hours and largely contains the yeast for up to 48 hours. Furthermore, the clear time-dependent leakage trend offers crucial experimental evidence and a defined time window for future optimization of the encapsulation efficacy.
