LNP Encapsulation
VectorBuilder는 RNA 및 plasmid 전달을 위한 LNP (lipid nanoparticle) encapsulation을 제공합니다. VectorBuilder의 서비스는 encapsulation 효율이 높은 균질한 LNP를 생산하는데 탁월합니다. 또한 조직-타겟 항체를 LNP에 접합하거나 LNP formulation을 최적화하여 약물 전달 효율성과 조직 타겟팅을 향상시킬 수 있습니다. LNP-RNA 치료제의 대량 제조에 대해서는 CDMO 서비스를 확인하세요.
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중점 사항
- 표준 (예: SM102, ALC-0315, MC3) 및 커스텀 formulation 가능
- mRNA, saRNA, siRNA, Cas9 mRNA/sgRNA mix, circRNA, pDNA 등 다양한 유형의 RNA/DNA 분자를 encapsulation 가능
- 높은 encapsulation 효율 (최대 100%)
- 낮은 (<0.1) PDI (polydispersity index)
- Antibody-conjugated LNP 가능
IVT Vector
Design & Cloning
Design & Cloning
IVT RNA Production
LNP Encapsulation Quality Control (QC) Functional Validation자세한 내용은 Therapeutic IVT RNA 개발 페이지를 참조하거나 Premade LNP-RNA 제품을 사용해 보세요.
품질관리 (QC)
VectorBuilder는 LNP encapsulated RNA 및 plasmid에 대한 종합적인 QC를 제공합니다.
| Attribute | QC assay | Research-grade | GMP-like |
|---|---|---|---|
| Appearance | Visual inspection | √ | √ |
| Concentration | RiboGreen assay | √ | √ |
| Encapsulation efficiency | RiboGreen assay | √ | √ |
| Particle size | Dynamic light scattering (Zetasizer) | √ | √ |
| Polydispersity index (PDI) | Dynamic light scattering (Zetasizer) | √ | √ |
| Surface charge (Zeta potential) | Dynamic light scattering (Zetasizer) | √ | √ |
| Encapsulated RNA integrity | Capillary gel electrophoresis (CGE) | Optional | √ |
| Endotoxin | Kinetic chromogenic assay (KCA) | Optional | √ |
| pH | pH paper | Optional | √ |
| Sterility | Bioburden test | Optional | √ |
LNP-mRNA QC 데이터
- TEM
- PDI and zeta potential
- LNP cryopreservation
Figure 1. Cryo-TEM micrographs of LNP-mRNA. Scale bar=100 nm.
Figure 2. Particle size and Zeta potential distribution analysis. PDI (A) and Zeta potential (B) were determined by DLS which measures the intensity differences of fluctuated light due to motion of particles. Data demonstrates homogeneous LNP mixtures.
Figure 3. Long-term (153 days) cryopreservation of anti-CD31 antibody-conjugated LNP-mRNA. (A) Firefly luciferase (FLuc)-expressing LNP-encapsulated mRNA was preserved under two conditions: 4 °C with no additives, and -80 °C in 7.5% sucrose solution. Particle size (pink bars) and PDI (deep cyan dots) of both groups were compared to the original parameters. (B) Imaging of Fluc mRNA expression 6 hours post-injection. Female ICR mice were intravenously injected with PBS, LNPs preserved at 4 °C without additives, or LNPs preserved at -80 °C in 7.5% sucrose solution. (C) Encapsulation efficiency of LNP-mRNA after cryopreservation. Freshly prepared FLuc LNP-mRNA was used as the control. (D) Comparison of RNA integrity after cryopreservation. Overall, our data show that storing antibody-conjugated LNP-mRNA at -80 °C in 7.5% sucrose solution effectively maintains particle homogeneity, encapsulation efficiency, and mRNA expression for long-term preservation.
LNP-RNA 기능 검증
- LNP-mRNA expression in vitro
- LNP-mRNA expression in vivo
- Primary T-Cell LNP Transfection
Figure 4. Efficient mRNA delivery and expression using LNP in vitro. Cells were transfected with LNP encapsulated EGFP mRNA or EGFP mRNA mixed with commercial transfection reagent. (A) Flow cytometry analysis of EGFP expression in Jurkat and 293T cells and (B) Fluorescent imaging of HEK293T cells at 24 hours post-transfection. MFI: median fluorescence intensity.
Figure 5. Expression of luciferase (Luc) mRNA and mRNA induced immune response in mice. (A) Luciferase activity visualized by live imaging at 6 h, 24 h, and 48 h post-injection. (B) Two pro-inflammatory cytokines, IL-6 and TNF-⍺, were quantified in the serum at 48 h post-injection. Error bars represent standard errors. Mice strain: C57BL/6J; mice age: 8 weeks; injection method: intramuscular injection. (C) IFN-γ ELISpot assay of splenocytes derived from Balb/C mice 14 days post intramuscular injection of 30 ug LNP-encapsulated mRNA coding for viral antigen A, viral antigen B, or control PBS.
Figure 6. Transfection of primary T-Cells with EGFP mRNA. (A) Histogram count of cells expressing EGFP as measured by flow cytometry 24 hours after transfection. Primary T-Cells were subjected to three different treatments: no treatment control (NC), mRNA transfected with a commercial transfection reagent, and lipid nanoparticle encapsulated mRNA (LNP). (B) Corresponding quantification of Median Fluorescence Intensity (black) and the % of EGFP-positive cells (grey) as measured by flow cytometry.
LNP Optimization
- Antibody-conjugated LNP
- Formulation optimization
- Novel formulation
- Tissue-targeting LNP
Figure 7. Anti-CD31 conjugated firefly luciferase (FLuc) LNP-mRNA showed improved luciferase expression in lung. Mice strain: C57BL/6J; mice age: 6-8 weeks; mice gender: female; administration route: tail vein. Negative controls: IgG2a-conjugated FLuc LNP-mRNA and naked FLuc mRNA.
Figure 8. LNP formulations achieve efficient plasmid transfection in vitro. (A) A plasmid overexpressing EGFP (pDNA-CMV>EGFP) was encapsulated with SM102 or ALC-0315 LNP and transfected to HEK293T cells. The EGFP expression was captured 24 h post-transfection. (B) Optimized SM102-based LNP formulation achieved ~6.6 times better transfection to HEK293T cells than a commercial PEI transfection reagent. A firefly luciferase expressing plasmid (pDNA-CMV>Fluc) was used as the reporter.
Figure 9. A Luciferase expressing plasmid (pDNA-CAG>Luc+) was encapsulated with VectorBuilder’s novel LNP formulation and was intravenously injected at 0.6 mg/kg body weight. Luciferase expression was observed till 96 h after injection.
Figure 10. Antibody conjugated LNP (lipid nanoparticles)를 사용한 조직 특이적 mRNA 전달. (A) Cre 의존 tdTomato 발현 카세트를 보유한 Ai9 마우스에 anti-CD31 antibody (0.4 mg/kg)와 결합하거나 결합하지 않은 LNP에 캡슐화된 Cre mRNA를 i.v. 주사했습니다. (B) 폐에서 tdTomato 발현을 주사 후 72시간에 시각화하였습니다.
주요 인용 논문
Phase Separation Enhanced PROTAC for Highly Efficient Protein Degradation
Yu, et al.
Biomacromolecules, 2024, doi: 10.1021/acs.biomac.4c00424
Products used:
Abstract: Biomacromolecular condensates formed via phase separation establish compartments for the enrichment of specific compositions, which is also used as a biological tool to enhance molecule condensation, thereby increasing the efficiency of biological processes. Proteolysis-targeting chimeras (PROTACs) have been developed as powerful tools for targeted protein degradation in cells, offering a promising approach for therapies for different diseases. Herein, we introduce an intrinsically disordered region in the PROTAC (denoted PSETAC), which led to the formation of droplets of target proteins in the cells and increased degradation efficiency compared with PROTAC without phase separation. Further, using a nucleus targeting intrinsically disordered domain, the PSETAC was able to target and degrade nuclear-located proteins. Finally, we demonstrated intracellular delivery of PSETAC using lipid nanoparticle-encapsulated mRNA (mRNA-LNP) for the degradation of the endogenous target protein. This study established the PSETAC mRNA-LNP method as a potentially translatable, safe therapeutic strategy for the development of clinical applications based on PROTAC.
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