Ko, J. ; Wang, Y. ; Sheng, K. ; Weitz, D. A. ; Weissleder, R. Sequencing-Based Protein Analysis of Single Extracellular Vesicles. ACS Nano 2021, 15, 5631-5638. Publisher's VersionAbstract

Circulating extracellular vesicles (EVs)—biological nanomaterials shed from most mammalian cells—have emerged as promising biomarkers, drug delivery vesicles, and treatment modulators. While different types of vesicles are being explored for these applications, it is becoming clear that human EVs are quite heterogeneous even in homogeneous or monoclonal cell populations. Since it is the surface EV protein composition that will largely dictate their biological behavior, high-throughput single EV profiling methods are needed to better define EV subpopulations. Here, we present an antibody-based immunosequencing method that allows multiplexed measurement of protein molecules from individual nanometer-sized EVs. We use droplet microfluidics to compartmentalize and barcode individual EVs. The barcodes/antibody-DNA are then sequenced to determine protein composition. Using this highly sensitive technology, we detected specific proteins at the single EV level. We expect that this technology can be further adapted for multiplexed protein analysis of any nanoparticle.

Xia, J. ; Cai, L. ; Wu, H. ; Mackintosh, F. C. ; Weitz, D. A. Anomalous mechanics of Zn2+-modified fibrin networks. PNAS 2021, 118. Publisher's VersionAbstract

Fibrin is the main component of blood clots. The mechanical properties of fibrin are therefore of critical importance in successful hemostasis. One of the divalent cations released by platelets during hemostasis is Zn2+; however, its effect on the network structure of fibrin gels and on the resultant mechanical properties remains poorly understood. Here, by combining mechanical measurements with three-dimensional confocal microscopy imaging, we show that Zn2+ can tune the fibrin network structure and alter its mechanical properties. In the presence of Zn2+, fibrin protofibrils form large bundles that cause a coarsening of the fibrin network due to an increase in fiber diameter and reduction of the total fiber length. We further show that the protofibrils in these bundles are loosely coupled to one another, which results in a decrease of the elastic modulus with increasing Zn2+ concentrations. We explore the elastic properties of these networks at both low and high stress: At low stress, the elasticity originates from pulling the thermal slack out of the network, and this is consistent with the thermal bending of the fibers. By contrast, at high stress, the elasticity exhibits a common master curve consistent with the stretching of individual protofibrils. These results show that the mechanics of a fibrin network are closely correlated with its microscopic structure and inform our understanding of the structure and physical mechanisms leading to defective or excessive clot stiffness.

An, P. ; Cantalupo, P. G. ; Zheng, W. ; Saenz-Robles, M. T. ; Duray, A. M. ; Weitz, D. A. ; Pipas, J. M. Single-Cell Transcriptomics Reveals a Heterogeneous Cellular Response to BK Virus Infection. Journal of Virology 2021, 95. Publisher's VersionAbstract

BK virus (BKV) is a human polyomavirus that is generally harmless but can cause devastating disease in immunosuppressed individuals. BKV infection of renal cells is a common problem for kidney transplant patients undergoing immunosuppressive therapy. In cultured primary human renal proximal tubule epithelial (RPTE) cells, BKV undergoes a productive infection. The BKV-encoded large T antigen (LT) induces cell cycle entry, resulting in the upregulation of numerous genes associated with cell proliferation. Consistently, microarray and transcriptome sequencing (RNA-seq) experiments performed on bulk infected cell populations identified several proliferation-related pathways that are upregulated by BKV. These studies revealed few genes that are downregulated. In this study, we analyzed viral and cellular transcripts in single mock- or BKV-infected cells. We found that the levels of viral mRNAs vary widely among infected cells, resulting in different levels of LT and viral capsid protein expression. Cells expressing the highest levels of viral transcripts account for approximately 20% of the culture and have a gene expression pattern that is distinct from that of cells expressing lower levels of viral mRNAs. Surprisingly, cells expressing low levels of viral mRNA do not progress with time to high expression, suggesting that the two cellular responses are determined prior to or shortly following infection. Finally, comparison of cellular gene expression patterns of cells expressing high levels of viral mRNA with those of mock-infected cells or cells expressing low levels of viral mRNA revealed previously unidentified pathways that are downregulated by BKV. Among these are pathways associated with drug metabolism and detoxification, tumor necrosis factor (TNF) signaling, energy metabolism, and translation.

Tang, J. ; Fan, B. ; Xiao, L. ; Tian, S. ; Zhang, F. ; Zhang, L. ; Weitz, D. A. A New Ensemble Machine-Learning Framework for Searching Sweet Spots in Shale Reservoirs. SPE Journal 2021, 26, 482-497. Publisher's VersionAbstract
Knowing the location of sweet spots benefits the horizontal well drilling and the selection of perforation clusters. Generally, geoscientists determine sweet spots from the well-logging interpretation. In this paper, a group of prevalent classifiers [extreme gradient boosting (XGBoost), unbiased boosting with categorical features (CatBoost), and light gradient boosting machine (LightGBM)] based on gradient-boosting decision trees (GBDTs) are introduced to automatically determine sweet spots based on well-log data sets. Compared with linear support vector machines (SVMs), these robust algorithms can deal with comparative scales of features and learn nonlinear decision boundaries. Moreover, they are less influenced by the presence of outliers. Another prevailing approach, named generative adversarial networks (GANs), is implemented to augment the training data set by using a small number of training samples. An extensive application has been built for the field cases in a certain oilfield. We randomly select 73 horizontal wells for training, and 13 features are chosen from well-log data sets. Compared with conventional SVMs, the agreement rates of interpretation by XGBoost and CatBoost are significantly improved. Without special preprocessing of the input data sets and conditional tabular GAN (CTGAN) model fine tuning, the fake data set could still bring a relatively low agreement rate for all detections. Finally, we propose an ensemble-learning framework concatenating multilevels of classifiers and improve agreement rate. In this paper, we illustrate a new tool for categorizing the reservoir quality by using GBDTs and ensemble models, which further helps search and identify sweet spots automatically. This tool enables us to integrate experts’ knowledge to the developed model, identify logging curves more efficiently, and cover more sweet spots during the drilling and completion treatment, which immensely decrease the cost of log interpretation.
Werner, J. G. ; Lee, H. ; Wiesner, U. ; Weitz, D. A. Ordered Mesoporous Microcapsules from Double Emulsion Confined Block Copolymer Self-Assembly. ACS Nano 2021, 15, 3490-3499. Publisher's VersionAbstract

Polymeric microcapsules with shells containing homogeneous pores with uniform diameter on the nanometer scale are reported. The mesoporous microcapsules are obtained from confined self-assembly of amphiphilic block copolymers with a selective porogen in the shell of water-in-oil-in-water double emulsion drops. The use of double emulsion drops as a liquid template enables the formation of homogeneous capsules of 100s of microns in diameter, with aqueous cores encapsulated in a shell membrane with a tunable thickness of 100s of nanometers to 10s of microns. Microcapsules with shells that exhibit an ordered gyroidal morphology and three-dimensionally connected mesopores are obtained from the triblock terpolymer poly(isoprene)-block-poly(styrene)-block-poly(4-vinylpyridine) coassembled with pentadecylphenol as a porogen. The bicontinuous shell morphology yields nanoporous paths connecting the inside to the outside of the microcapsule after porogen removal; by contrast, one-dimensional hexagonally packed cylindrical pores, obtained from a traditional diblock copolymer system with parallel alignment to the surface, would block transport through the shell. To enable the mesoporous microcapsules to withstand harsh conditions, such as exposure to organic solvents, without rupture of the shell, we develop a cross-linking method of the nanostructured triblock terpolymer shell after its self-assembly. The microcapsules exhibit pH-responsive permeability to polymeric solutes, demonstrating their potential as a filtration medium for actively tunable macromolecular separation and purification. Furthermore, we report a tunable dual-phase separation method to fabricate microcapsules with hierarchically porous shells that exhibit ordered mesoporous membrane walls within sponge-like micron-sized macropores to further control shell permeability.

Tiribocchi, A. ; Montessori, A. ; Lauricella, M. ; Bonaccorso, F. ; Succi, S. ; Aime, S. ; Milani, M. ; Weitz, D. A. The vortex-driven dynamics of droplets within droplets. Nature Communications 2021, 12, 1-10. Publisher's VersionAbstract

Understanding the fluid-structure interaction is crucial for an optimal design and manufacturing of soft mesoscale materials. Multi-core emulsions are a class of soft fluids assembled from cluster configurations of deformable oil-water double droplets (cores), often employed as building-blocks for the realisation of devices of interest in bio-technology, such as drug-delivery, tissue engineering and regenerative medicine. Here, we study the physics of multi-core emulsions flowing in microfluidic channels and report numerical evidence of a surprisingly rich variety of driven non-equilibrium states (NES), whose formation is caused by a dipolar fluid vortex triggered by the sheared structure of the flow carrier within the microchannel. The observed dynamic regimes range from long-lived NES at low core-area fraction, characterised by a planetary-like motion of the internal drops, to short-lived ones at high core-area fraction, in which a pre-chaotic motion results from multi-body collisions of inner drops, as combined with self-consistent hydrodynamic interactions. The onset of pre-chaotic behavior is marked by transitions of the cores from one vortex to another, a process that we interpret as manifestations of the system to maximize its entropy by filling voids, as they arise dynamically within the capsule.

Ko, J. ; Wang, Y. ; Carlson, J. C. T. ; Marquard, A. ; Gungabeesoon, J. ; Charest, A. ; Weitz, D. A. ; Pittet, M. J. ; Weissleder, R. Single Extracellular Vesicle Protein Analysis Using Immuno-Digital Droplet Polymerase Chain Reaction Amplification. Advanced Biosystems 2020, 4 1900307. Publisher's VersionAbstract

There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno‐droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody–DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read‐out by droplet imaging. In these proof‐of‐principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.

Ding, R. ; Hung, K. - C. ; Mitra, A. ; Ung, L. W. ; Lightwood, D. ; Tu, R. ; Starkie, D. ; Cai, L. ; Mazutis, L. ; Chong, S. ; et al. Rapid isolation of antigen-specific B-cells using droplet microfluidics. RSC Advances 2020, 10, 27006-27013. Publisher's VersionAbstract
Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusion inefficiency and display bias, respectively. Here we present a platform, featuring droplet microfluidics and a bead-based binding assay, to rapidly identify and verify antigen-binding antibody sequences from primary cells. We used a defined mixture of hybridoma cells to characterize the system, sorting droplets at up to 100 Hz and isolating desired hybridoma cells, comprising 0.1% of the input, with a false positive rate of less than 1%. We then applied the system to once-frozen primary B-cells to isolate rare cells secreting target-binding antibody. We performed RT-PCR on individual sorted cells to recover the correctly paired heavy- and light-chain antibody sequences, and we used rapid cell-free protein synthesis to generate single-chain variable fragment-format (scFv) antibodies from fourteen of the sorted cells. Twelve of these showed antigen-specific binding by ELISA. Our platform facilitates screening animal B-cell repertoires within days at low cost, increasing both rate and range of discovering antigen-specific antibodies from living organisms. Further, these techniques can be adapted to isolate cells based on virtually any secreted product.
Jeong, H. - S. ; Kim, E. ; Nam, C. ; Choi, Y. ; Lee, Y. - J. ; Weitz, D. A. ; Lee, H. ; Choi, C. - H. Hydrogel Microcapsules with a Thin Oil Layer: Smart Triggered Release via Diverse Stimuli. Advanced Functional Materials 2020. Publisher's VersionAbstract

A hydrogel microcapsule with an intermediate thin oil layer is presented to achieve smart release of a broad range of cargoes triggered via diverse stimuli. A microfluidic technique is used to produce triple emulsion droplets with a thin oil layer that separates the innermost aqueous phase from the hydrogel prepolymer phase, which transforms into a hydrogel shell via photopolymerization. The intermediate oil layer within the hydrogel microcapsule acts as an effective diffusion barrier, allowing encapsulation of various small cargoes within a porous hydrogel shell until a stimulus is applied to destabilize the oil layer. It is demonstrated that diverse stimuli including chemical dissolution, mechanical stress, and osmotic pressure can be utilized to release the encapsulated cargo on‐demand. In addition, osmotic pressure and the hydrogel shell thickness can be independently tuned to control the onset time of release as well as the release behavior of multi‐cargo encapsulated hydrogel microcapsule. The release can be either simultaneous or selective.

Sabbagh, B. ; Stolovicki, E. ; Park, S. ; Weitz, D. A. ; Yossifon, G. Tunable Nanochannels Connected in Series for Dynamic Control of Multiple Concentration-Polarization Layers and Preconcentrated Molecule Plugs. Nano Letters 2020, 20, 8524-8533. Publisher's VersionAbstract
Integration of ionic permselective medium (e.g., nanochannels, membranes) within microfluidic channels has been shown to enable on-chip desalination, sample purification, bioparticle sorting, and biomolecule concentration for enhanced detection sensitivity. However, the ion-permselective mediums are generally of fixed properties and cannot be dynamically tuned. Here we study a microfluidic device consisting of an array of individually addressable elastic membranes connected in series on top of a single microfluidic channel that can be deformed to locally reduce the channel cross-section into a nanochannel. Dynamic tunability of the ion-permselective medium, as well as controllability of its location and ionic permselectivity, introduces a new functionality to microfluidics-based lab-on-a-chip devices, for example, dynamic localization of preconcentrated biomolecule plugs at different sensing regions for multiplex detection. Moreover, the ability to simultaneously form a series of preconcentrated plugs at desired locations increases parallelization of the system and the trapping efficiency of target analytes.
Wu, Z. ; Werner, J. G. ; Weitz, D. A. Microfluidic fabrication of Phase-Inverted Microcapsules with Asymmetric Shell Membranes with Graded Porosity. ACS Macro Letters 2020, 10, 116-121. Publisher's VersionAbstract
Microcapsules with liquid cores and solid shells are attractive as dispersible protective micron-sized containers. Applications that rely on molecular mass transport often require a combination of size selectivity, high permeability, and mechanical stability. Capsule architectures that combine all these features represent a material property, design, and fabrication challenge. In this work, the design of an asymmetric microcapsule shell architecture is reported to achieve a good combination of the desired features. Poly(methyl methacrylate) phase-inverted microcapsules featuring an asymmetric graded macroporous shell covered with a dense skin separation layer are obtained from water-in-oil-in-water double emulsion drops that are phase-inverted in a water-based coagulation bath. The phase-inverted microcapsules exhibit good mechanical stability and allow for high permeability of its shell membrane with molecular size dependence.
Hwang, H. ; Weitz, D. A. ; Spaepen, F. Stiffness of the interface between a colloidal body-centered cubic crystal and its liquid. PNAS 2020, 117, 25225-25229. Publisher's VersionAbstract
Equilibrium interfaces were established between body-centered cubic (BCC) crystals and their liquid using charged colloidal particles in an electric bottle. By measuring a time series of interfacial positions and computing the average power spectrum, their interfacial stiffness was determined according to the capillary fluctuation method. For the (100) and the (114) interfaces, the stiffnesses were 0.15 and 0.18 kBT/σ2 (σ: particle diameter), respectively, and were isotropic in the plane of the interface. For comparison, similar charged colloids were used to create an interface between a face-centered cubic (FCC) crystal and its liquid. Its stiffness was significantly larger: 0.26 kBT/σ2. This result gives experimental support to the explanations offered for the preferential nucleation of BCC over FCC in metallic alloys.
Brenner, M. ; Sorensen, P. ; Weitz, D. Science and Cooking: Physics Meets Food, From Homemade to Haute Cuisine; 1st ed. W.W. Norton, 2020; pp. 320. Publisher's Version
Sun, Z. ; Wu, B. ; Ren, Y. ; Wang, Z. ; Zhao, C. ‐X. ; Hai, M. ; Weitz, D. A. ; Chen, D. Diverse Particle Carriers Prepared by Co-Precipitation and Phase Separation: Formation and Applications. ChemPlusChem 2020, 86, 49-58. Publisher's VersionAbstract
Nanoparticles with diverse structures and unique properties have attracted increasing attention for their widespread applications. Co‐precipitation under rapid mixing is an effective method to obtained biocompatible nanoparticles and diverse particle carriers are achieved by controlled phase separation via interfacial tensions. In this Minireview, we summarize the underlying mechanism of co‐precipitation and show that rapid mixing is important to ensure co‐precipitation. In the binary polymer system, the particles can form four different morphologies, including occluded particle, core‐shell capsule, dimer particle, and heteroaggregate, and we demonstrate that the final morphology could be controlled by surface tensions through surfactant, polymer composition, molecular weight, and temperature. The applications of occluded particles, core‐shell capsules and dimer particles prepared by co‐precipitation or microfluidics upon the regulation of interfacial tensions are discussed in detail, and show great potential in the areas of functional materials, colloidal surfactants, drug delivery, nanomedicine, bio‐imaging, displays, and cargo encapsulation.
Liu, Y. ; Yang, G. ; Jin, S. ; Zhang, R. ; Chen, P. ; Tengjisi, ; Wang, L. ; Chen, D. ; Weitz, D. A. ; Zhao, C. ‐X. J-Aggregate-Based FRET Monitoring of Drug Release from Polymer Nanoparticles with High Drug Loading. Angewandte Chemie 2020, 59, 20065-20074. Publisher's VersionAbstract
Understanding drug‐release kinetics is critical for the development of drug‐loaded nanoparticles. We developed a J‐aggregate‐based Förster‐resonance energy‐transfer (FRET) method to investigate the release of novel high‐drug‐loading (50 wt %) nanoparticles in comparison with low‐drug‐loading (0.5 wt %) nanoparticles. Single‐dye‐loaded nanoparticles form J‐aggregates because of the high dye‐loading (50 wt %), resulting in a large red‐shift (≈110 nm) in the fluorescence spectrum. Dual‐dye‐loaded nanoparticles with high dye‐loading using FRET pairs exhibited not only FRET but also a J‐aggregate red‐shift (116 nm). Using this J‐aggregate‐based FRET method, dye‐core–polymer‐shell nanoparticles showed two release processes intracellularly: the dissolution of the dye aggregates into dye molecules and the release of the dye molecules from the polymer shell. Also, the high‐dye‐loading nanoparticles (50 wt %) exhibited a slow release kinetics in serum and relatively quick release in cells, demonstrating their great potential in drug delivery.
Lin, S. ‐Z. ; Chen, P. ‐C. ; Guan, L. ‐Y. ; Shao, Y. ; Hao, Y. - K. ; Li, Q. ; Li, B. ; Weitz, D. A. ; Feng, X. ‐Q. Universal Statistical Laws for the Velocities of Collective Migrating Cells. Advanced Biosystems 2020, 4 2000065. Publisher's VersionAbstract
Migratory dynamics of collective cells is central to the morphogenesis of biological tissues. The statistical distribution of cell velocities in 2D confluent monolayers is measured through large‐scale and long‐term experiments of various cell types lying on different substrates. A linear relation is discovered between the variability and the mean of cell speeds during the jamming process of confluent cell monolayers, suggesting time‐invariant distribution profile of cell velocities. It is further found that the probability density function of cell velocities obeys the non‐canonical q‐Gaussian statistics, regardless of cell types and substrate stiffness. It is the Tsallis entropy, instead of the classical Boltzmann–Gibbs entropy, that dictates the universal statistical laws of collective cell migration. The universal statistical law stems from cell–cell interactions, as demonstrated by the wound healing experiments. This previously unappreciated finding provides a linkage between cell‐level heterogeneity and tissue‐level ensembles in embryonic development and tumor growth.
Gerber, G. ; Weitz, D. A. ; Coussot, P. Propagation and adsorption of nanoparticles in porous medium as traveling waves. Physical Review Research 2020, 2 033074. Publisher's VersionAbstract

Generally, one attempts to globally predict or interpret, from numerical simulations, the history of the effluents exiting porous media (i.e., the breakthrough curve), without a clear view of the detailed evolutions of deposition inside the medium. We developed a simple physical frame of description of the colloidal particle transport and adsorption, which allows to predict the main characteristics of transport and deposition in porous media from a set of directly measurable (macroscopic) physical parameters. More precisely, we show that the deposition distribution is basically a traveling wave propagating in the medium with a shape (frontal or extended) and velocity depending on the flow rate and the availability of particles with regards to the adsorption capacity. This in particular makes it possible to predict or interpret the breakthrough curve shape from a physical approach. We also show that additional effects may be included, such as a multiporosity leading to confinement effects (delayed deposition in less accessible regions). The validity of the model is checked from original direct visualizations by confocal microscopy of particle adsorption in time and space for nanoparticle suspensions flowing through a bead packing. This makes it possible to measure the evolution of the deposition profiles in time distinguishing the deposition in confined regions. The model appears to successfully predict the different trends: traveling wave, global deposition profile shape, profiles of deposition in confined regions.

    Zhang, L. ; Abbaspourrad, A. ; Parsa, S. ; Tang, J. ; Cassiola, F. ; Zhang, M. ; Tian, S. ; Dai, C. ; Xiao, L. ; Weitz, D. A. Core-Shell Nanohydrogels with Programmable Swelling for Conformance Control in Porous Media. ACS Applied Materials & Interfaces 2020, 12, 34217-34225. Publisher's VersionAbstract

    Conformance control during waterflooding in an oil reservoir is utilized to redistribute water and increase the sweep efficiency and hence oil production. Using preformed gel particles can effectively redirect the flow by blocking the high-permeability zones and forcing water into low-permeability zones where the oil is trapped. However, the size of such gel particles can limit their applications deeper within the reservoir and can result in shear-induced degradation near the well bore. Here, we fabricate core–shell nanohydrogels with delayed swelling behavior; their volume increases by a factor of 200 after about 30 days in brine under reservoir conditions. We study their effect on the flow behavior in a three-dimensional porous medium micromodel consisting of randomly packed glass beads. Using confocal microscopy, we directly visualize the spatial variations of flow in the micromodel before and after nanohydrogel injection and swelling. The swollen nanohydrogels block some pores reducing the permeability of the micromodel and diverting the water into low-permeability regions. A core flood experiment further confirms that the nanohydrogels can significantly reduce the permeability of a reservoir sample and divert the fluid flow. Our results demonstrate that these core–shell nanohydrogels might be useful for flow control in porous media and can be used as a conformance control agent.

    Wang, Y. ; Cao, T. ; Ko, J. ; Shen, Y. ; Zong, W. ; Sheng, K. ; Cao, W. ; Sun, S. ; Cai, L. ; Zhou, Y. ‐L. ; et al. Dissolvable Polyacrylamide Beads for High-Throughput Droplet DNA Barcoding. Advanced Science 2020, 7 1903463. Publisher's VersionAbstract
    Droplet‐based single cell sequencing technologies, such as inDrop, Drop‐seq, and 10X Genomics, are catalyzing a revolution in the understanding of biology. Barcoding beads are key components for these technologies. What is limiting today are barcoding beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency. Here, this work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers. The dissolvable beads are easy to synthesize, and the primer cost for the beads is significantly lower than that for the previous barcoding beads. Furthermore, the dissolvable beads can be loaded into drops with >95% loading efficiency of a single bead per drop and the dissolution of beads does not influence reverse transcription or the polymerase chain reaction (PCR) in drops. Based on this approach, the dissolvable beads are used for single cell RNA and protein analysis.
    Liu, Y. ; Yang, G. ; Baby, T. ; Tengjisi, ; Chen, D. ; Weitz, D. A. ; Zhao, C. ‐X. Stable Polymer Nanoparticles with Exceptionally High Drug Loading by Sequential Nanoprecipitation. Angewandte Chemie 2020, 59, 4720-4728. Publisher's VersionAbstract
    Poor solubility often leads to low drug efficacy. Encapsulation of water‐insoluble drugs in polymeric nanoparticles offers a solution. However, low drug loading remains a critical challenge. Now, a simple and robust sequential nanoprecipitation technology is used to produce stable drug‐core polymer‐shell nanoparticles with high drug loading (up to 58.5 %) from a wide range of polymers and drugs. This technology is based on tuning the precipitation time of drugs and polymers using a solvent system comprising multiple organic solvents, which allows the formation of drug nanoparticles first followed by immediate precipitation of one or two polymers. This technology offers a new strategy to manufacture polymeric nanoparticles with high drug loading having good long‐term stability and programmed release and opens a unique opportunity for drug delivery applications.