Application Note

纳米发电机™纳米粒子合成系统

PreciGenome NanoGenerator™ 是用于纳米粒子合成的高性能仪器，如脂质纳米粒子、脂质体、PLGA 等，广泛用于药物输送、基因治疗、LNP 配制和制造等。

NanoGenerator™ 生成的纳米粒子具有更好的尺寸均匀性和更小的 PDI。它可从 0.1mL/样品筛选扩展到 1L（>10L 定制设计）大批量 GMP 生产

纳米粒子，尤其是脂质体和聚合物纳米粒子，由于其优异的性能，在药物递送、mRNA疫苗和生物传感等制药工业等各个领域显示出巨大的生物医学应用潜力。

通过微流控技术合成纳米颗粒比传统的批量合成工艺具有优势，因为它能够在尺寸和形状上具有更好的均匀性。例如，在药物递送领域，使用NanoGenerator™纳米粒子合成系统可以合成脂质纳米粒子（LNP）、脂质体、PLGA等多种纳米粒子。脂质纳米颗粒 (LNP)、脂质体和 PLGA 是最常用的可生物降解材料，用于输送亲水性和疏水性化合物。

1. Lipid Nanoparticle Synthesis and Manufacturing

Lipid nanoparticles are versatile delivery systems composed of lipid bilayers capable of encapsulating various therapeutic agents. These nanocarriers have gained prominence for delivering a diverse array of therapeutics, particularly biological products like proteins and nucleic acids [1]. The fundamental composition of LNPs typically includes four primary components: cationic or ionizable lipids that bind with negatively charged biological material and assist in endosomal escape; phospholipids that provide structural integrity; cholesterol that contributes to stability and enables membrane fusion; and PEGylated lipids that improve stability and circulation [1].

The choice of formulation method is crucial as it directly impacts nearly all critical quality attributes (CQAs) of the final product, including particle size, encapsulation efficiency, yield, and morphology. These in turn ultimately determine pharmacokinetics, cellular uptake, and overall efficacy. Therefore, understanding synthesis and manufacturing methods is essential for developing effective LNP-based therapeutics. Additionally, the primary challenges faced at each stage of production will differ, so it is important to consider this when determining which production method is most appropriate.

1. LNP Synthesis and Manufacturing

1.1 Drug development process steps and requirements

LNP drug development starts with formulation screening, where several combinations of payload, lipid mix, and synthesis conditions are tested for optimal performance. At this stage, priority is placed on rapid production of small batches of LNPs. This permits lower cost, higher throughput screening for the most promising leads. As a result, ideal production methods permit reliable and repeatable results even with low production volumes.

At later stages of production, priorities shift towards stable continuous LNP synthesis due to fewer formulations left to test. With production volumes shifting from the hundreds of microliters to milliliters or even liters, the ideal synthesis method must be practical for larger volumes while maintaining scalability with findings from earlier production stages. The latter quality is especially important, as this can save valuable time by forgoing the need to design brand new experimental protocols.

For the final stages where the drug is ready for human trials, additional care must be taken to follow current good manufacturing practices (cGMP) so that the product is appropriate for release on the market. As a result, the production method should not only permit more continuous LNP synthesis, it must also be compatible with workflows that strictly follow pharmaceutical guidelines.

Fig 1. Phases of LNP drug development

1.2 Classification of Traditional LNP Synthesis Methods

Traditional LNP manufacturing methods can be broadly categorized into two main classes:

Top-down methods: Also referred to as high-energy methods, these involve applying energy to break larger particles into smaller ones [2].
Bottom-up methods: These rely on the self-assembly of lipid components to form homogeneous nanoparticle systems[2].

Additionally, synthesis approaches can be further classified as:

High-energy methods
Low-energy methods
Organic solvent-based methods [1]

1.2.1 Top-Down Synthesis Methods

Sonication

Sonication is one of the most widespread methods for synthesizing liposomes (Fig.2) [3]. The technique utilizes ultrasound to shrink droplets to the desired size and improve uniformity. After melting the lipid, it is dispersed in an aqueous phase with surfactant and rapidly mixed to generate emulsions. The ultrasound is then applied to minimize droplet size, followed by gradual cooling to result in LNPs.

This method is suitable for small-scale production and takes advantage of commonly available lab equipment. However, sonication has shortcomings with encapsulating payloads and can be imperfect at removing microparticles from the product.

Fig 2. Overview of ultrasonication. Image from Pharmaceutics 2024, 16:131.

High-Pressure Homogenization

High-pressure homogenization is primarily used to obtain small vesicles and reduce the size of multilamellar liposomes (Fig.3) [3]. Briefly, an emulsion of molten lipid with surfactant is pushed through a small orifice and accelerated at high pressure to break down into LNPs. This method may either be performed hot, where the emulsion is homogenized before cooling down, or cold, where the emulsion is homogenized after cooling down.

This method is ideal for incorporation of lipophilic drugs. However, high-pressure homogenization can the use of high temperatures can potentially cause degradation, even with rapid cooling.

Fig 3. Overview of high-pressure homogenization. Image from Pharmaceutics 2024, 16:131.

1.2.2 Bottom-Up Synthesis Methods

Membrane Extrusion

Membrane extrusion is a common industrial method for obtaining homogeneous liposomes by extruding the emulsion through a polycarbonate membrane with uniform pore size (Fig.4) [3]. This emulsion is then circulated under specific parameters like time, pressure, and temperature.

Fig 4. Overview of membrane extrusion. Image from Pharmaceutics 2024, 16:131.

Thin-Film Hydration

Thin-film hydration is the oldest and perhaps easiest method of LNP preparation (Fig.5) [5]. Briefly, an organic solvent containing lipid mix is dried down, forming a thin film. Hydrating this film then results in a liposomal dispersion, where more intense hydration results in more layers to the resulting vesicles. The resulting LNPs tend to have poor uniformity, so this method is often combined with membrane extrusion to improve homogeneity [3].

Fig 5. Overview of thin-film hydration. Image from Pharmaceutics 2024, 16:131.

Solvent-Evaporation

Solvent-evaporation (also known as emulsification or reverse-phase evaporation) is a prominent technique for liposome preparation that offers high encapsulation efficiency (Fig.6) [4]. In this method, lipid dissolved in an organic solvent is mixed with an aqueous buffer containing a drug of interest. The organic solvent is then evaporated, generating LNPs which may be resuspended in aqueous buffer.

Solvent-evaporation is ideal for producing LNPs with a high water-to-lipid ratio. Like thin-film hydration though, it must often be combined with another procedure to improve product uniformity.

Fig 6. Overview of solvent-evaporation. Image from Pharmaceutics 2024, 16:131.

Solvent Injection

Solvent injection (also known as ethanol injection) involves the rapid injection of a lipid-ethanol solution into an excess of buffer. This leads to immediate production of LNPs as the lipid precipitates (Fig.7) [3].

This method is simple to carry out, but has poor uniformity of LNPs. Additionally, mixing both phases results in both a dilute LNP solution, as well as one with a high ethanol concentration, which negatively affects LNP stability. Post-processing will therefore be required to both concentrate the LNPs and remove ethanol from the solution.

Fig 7. Overview of solvent injection. Image from Pharmaceutics 2024, 16:131.

1.3 Microfluidics vs. Traditional Methods

Solvent Injection

Solvent injection (also known as ethanol injection) involves the rapid injection of a lipid-ethanol solution into an excess of buffer. This leads to immediate production of LNPs as the lipid precipitates (Fig.7) [3].

This method is simple to carry out, but has poor uniformity of LNPs. Additionally, mixing both phases results in both a dilute LNP solution, as well as one with a high ethanol concentration, which negatively affects LNP stability. Post-processing will therefore be required to both concentrate the LNPs and remove ethanol from the solution.

Fig 8. Overview of microfluidic mixing

1.4 Comparison of Methods

Method	Advantages	Limitations	Encapsulation Efficiency	Scalability

Microfluidic mixing offers several advantages over traditional methods in key areas:

Size Homogeneity

Bulk mixing in traditional methods often results in polydisperse LNPs, which complicates biodistribution and therapeutic efficacy [6]. In contrast, controlled continuous flow offers stable flow rate conditions for monodisperse LNPs, with PDI below 0.2 [8].

Encapsulation Efficiency

Passive loading in techniques like film hydration leads to inconsistent encapsulation of nucleic acids or drugs, with efficiencies often below 50% [6][7]. This is not the case for microfluidics, where both phases are uniformly suspended before mixing in a microscale environment. Consequently, encapsulation efficiencies of over 95% are readily achievable [7][8].

Batch-to-Batch Consistency

Manual handling and uncontrolled mixing parameters introduce variability, making clinical translation challenging [6][8]. Microfluidic mixing however provides options for automated workflows with repeatable results.

Scalability

Scaling up batch processes requires complex optimization, often compromising nanoparticle quality [6]. With microfluidics however, scaling up may be done with the same mixing channel technology with relatively minor adjustments for higher throughput (Fig.9).

Fig 9. Scalability of LNP synthesis across different NanoGenerator Instruments. Equivalent performance is maintained across different throughput scales through adjustments to the same mixing channel technology.

1.5 Critical Parameters in Microfluidic LNP Synthesis

The high precision afforded by microfluidic synthesis is in turn dependent on several tunable parameters. Good repeatability requires these parameters to remain constant both within the same production batch and between separate batches.

Total Flow Rate (TFR)

TFR influences mixing kinetics and shear forces within microchannels. Higher TFRs enhance turbulence, reducing diffusion distances and ensuring homogeneous mixing. This results in smaller LNPs (70–100 nm) with narrower polydispersity (PDI <0.2) [9][12][13]. Conversely, low TFRs prolong mixing times, leading to heterogeneous size distributions and suboptimal encapsulation efficiency (Fig.10) [9][13].

Due to TFR's relationship with factors like fluidic resistance or mixing efficiency, it is important to remember these trends are relative. When comparing mixing channels with different properties, equal TFRs will not necessarily yield equivalent results. As a result, absolute TFR values are not as important in isolation.

Fig 10. Effects of TFR on the NanoGenerator Flex-M. Other synthesis parameters such as formulation and FRR were fixed.

Flow Rate Ratio (FRR)

FRR may describe the ratio of the aqueous phase flow rate to the organic phase flow rate, or vice versa. To avoid confusion, this post will refer exclusively to aqueous:organic FRRs.

Studies demonstrate that higher FRRs (e.g., 3:1 to 10:1) accelerate ethanol dilution, promoting rapid lipid self-assembly into smaller nanoparticles (Fig.11). For instance, at an FRR of 3:1, LNPs with diameters of ~100 nm and encapsulation efficiencies (EE) >80% are achievable, whereas lower FRRs (<2:1) yield larger particles (>150 nm) and reduced encapsulation efficiency due to insufficient mixing [9][10][11]. The abrupt drop in ethanol concentration at high FRRs limits lipid aggregation time, favoring nucleation over growth phases. However, too high of an FRR leads to a drop in LNP stability, so it is important to find an appropriate balance with this parameter.

Fig 11. Effects of FRR on the NanoGenerator Flex-M. Other synthesis parameters such as formulation and TFR were fixed.

N/P Ratio

The N/P ratio is the molar ratio of the amine (N) groups in the ionizable lipid to the phosphate (P) groups in the payload. This parameter affects encapsulation efficiency, particle size, and transfection potency by determining the electrostatic interactions between cationic lipids and anionic nucleic acids. Furthermore, it exhibits complex interdependent relationships with other paramters such as FRR and ionizable lipid content.

The optimal N/P ratio is payload-dependent, with an N/P ratio of approximately 3 commonly used for siRNA formulations and an N/P ratio of 6 typically employed for mRNA delivery systems [16]. This higher ratio for mRNA LNPs likely accommodates the larger molecular size and charge density of mRNA compared to siRNA, ensuring sufficient complexation while maintaining favorable nanoparticle characteristics.

While the N/P ratio is typically set as a target which determines required reagent concentrations, it is also possible to calculate the N/P ratio of a given mixture if the ionizable lipid mass; ionizable lipid molecular weight; amine groups per ionizable lipid molecule; and nucleic acid mass are known:

Lipid Concentration

Lipid concentration in the organic phase directly affects LNP size and stability. Lower concentrations produce smaller nanoparticles due to limited lipid availability for aggregation, while higher concentrations yield larger particles [11][15]. However, excessively low concentrations risk incomplete encapsulation, necessitating a balance between size and payload capacity. For example, 10 mg/mL lipid solutions optimized for siRNA delivery achieve 80–90% EE with minimal PDI, even though 5 mg/mL lipid solutions would yield smaller LNPs [11][15].

Micromixer Geometry

The design of the microfluidic mixer dictates mixing efficiency and fluidic resistance. Devices with chaotic advection features (e.g., staggered herringbone or toroidal mixers) induce transverse flow components, reducing diffusion lengths and enhancing homogeneity. For instance, chaotic mixers with 31 µm depths produce LNPs 20% smaller than those from simple T-junction designs [14][15]. As for fluidic resistance, while this does not directly affect LNP quality, it does affect the range of TFRs practically attainable with a given micromixer design.

Ethanol Concentration

Ethanol is the most common organic solvent used for the lipid phase in LNP synthesis and must be rapidly diluted to trigger nanoparticle formation. Microfluidic systems achieve critical ethanol concentrations (60–80%) within milliseconds, ensuring uniform nucleation. However, prolonged exposure to intermediate ethanol levels (30–50%) causes lipid precipitation and size heterogeneity [11], so it is important to reduce ethanol content post-synthesis (e.g. Inline dilution) to maintain LNP quality. Post-processing options such as tangential flow filtration (TFF) or diafiltration can reduce residual ethanol to <5% or less, enhancing colloidal stability [9][11].

Temperature

Temperature modulates lipid fluidity and mixing viscosity. Elevated temperatures reduce ethanol viscosity, accelerating dilution and favoring smaller LNPs [11][12]. However, excessive heat risks lipid oxidation, particularly for unsaturated variants like DOPC [14]. Additionally, these changes to viscosity can affect flow rate consistency if instrumentation is not properly calibrated to the given temperature range.

Parameter	Effect

2. Downstream Processing and Characterization of LNPs

Transitioning results from research to clinical applications requires robust downstream processing and characterization protocols. These procedures permit not only guided optimization of microfluidic synthesis parameters, but also quality assurance of the final LNP product.

2.1 Downstream Processing

Downstream processing encompasses purification, concentration, preservation, and fill/finish steps, each critical for optimizing LNP functionality and stability.

Purification

Purification removes impurities such as unencapsulated drugs, solvents, and excess lipids. Common methods include:

Tangential Flow Filtration (TFF): A continuous process using membranes to retain LNPs while removing solvents (e.g., ethanol) and free drugs. Studies demonstrate TFF achieves >95% ethanol removal and >90% drug purification in ≤4 minutes [17][18]. For cationic LNPs, TFF maintains 87–96% lipid recovery while reducing solvent residues to <5% [17].
Diafiltration: Exchanges buffers to stabilize LNPs. For example, replacing ethanol with phosphate-buffered saline (PBS) prevents lipid destabilization [19].

Concentration

Increasing concentration after production can improve LNP potency without altering particle size [18][20]. One method is with TFF-mediated volume reduction, which combines ultrafiltration and evaporation.

Preservation

Special procedures must be undertaken to preserve LNPs long-term. The most common method is lyophilization, or freeze-drying LNPs with cryoprotectants (e.g., 10–30% sucrose). Lyophilized mRNA-LNPs retain >95% activity after 12 weeks at 4°C [19][21].

Fill/Finish

Sterile filling into vials or syringes ensures product integrity. Automated systems minimize contamination risks, critical for GMP compliance [20].

2.2 Characterization of LNPs

Characterization verifies critical quality attributes (CQAs) and guides process optimization. Typical LNP CQAs are size and polydispersity; encapsulation efficiency; surface charge; and overall stability.

Particle Size and Polydispersity

Both size and polydispersity are most often measured with dynamic light scattering (DLS). Cryo-TEM may also be used to visualize LNP morphology, which is not reported by DLS. This can be useful to further characterize LNP structure [22][23][24].

Encapsulation Efficiency

Encapsulation efficiency may be measured with fluorescence assays. Using dyes (e.g. propidium iodide), these can quantify free payload to determine how much was encapsulated. An alternative is ion-pair reversed-phase chromatography (IP-RP), which can quantify both free and encapsulated nucleic acids [25][26].

Regardless of the method used to measure encapsulation efficiency, the principle behind calculating it is the same and goes by the following equation:

Surface Charge (Zeta Potential)

Electrophoretic light scattering (ELS) can be used to determine zeta potential, which is critical for predicting biodistribution. Cationic LNPs (ζ > +20 mV) exhibit hepatotoxicity, while anionic variants (ζ < −10 mV) avoid immune activation [22].

Stability

Long term stability tests may be conducted with lyophilization to ensure its effectiveness. This parameter is intertwined with the other previously mentioned CQAs, so example tests may include monitoring particle size and mRNA integrity post-reconstitution.

References:

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