Bead Loading Research Articles

Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1’s Behavior in Live Cells

AbstractUniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named “suspension bead loading” (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin‐related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

Stirred media mills of fine and ultrafine grinding for subsequent beneficiation operations

This paper presents a brief review of the designs, characteristics, and operating parameters of Stirred media mills supplied by foreign manufacturers. An analysis of information on Stirred media mills , which are used to improve the efficiency of beneficiation and metallurgical operations, is carried out using available publications. The obtained results indicate the inexpediency of using conventional drum ball mills for flotation separation of ores and concentrates with a fine interpenetration of minerals. The application of conventional mills in extraction of ores and concentrates resistant to cyanidation, where gold is finely disseminated in sulfides, is limited by their high power consumption and the corresponding operating costs. In this work, different types of Stirred media mills are considered, including their vertical and horizontal types. The advantages of Stirred media mills over conventional ball mills, particularly in terms of energy efficiency, are noted. The designs and main characteristics of Stirred media mills , which have received wide application, are presented. According to the data presented in the reviewed publications, the main parameters determining the grinding process in such mills are outlined, including the ratio of bead size to mill feed size, density of grinding medium, density of pulp in the mill, volume loading of beads, stirring speed, etc. The conducted analysis of literature data indicated high efficiency of mills based on agitation of grinding media for fine and ultrafine grinding of ores and concentrates in comparison with conventional ball mills. Given that the volume of ores and concentrates resistant to processing by conventional methods is increasing, the application of Stirred media mills is expected become more widespread, thereby increasing the efficiency of subsequent beneficiation and metallurgical operations.

Temperature mapping of milling by dual centrifugation: A systematic investigation

Using low quantities of drug compounds is often favorable in the early stages of drug development, especially for what require a large screening investigation to define the final formulation composition, such as nano- and microsuspensions. For that reason, the dual centrifugation approach has in the recent years been used due to its reproducible and fast-milling capacity with 40 samples in 2 mL vials simultaneously without the addition of cooling breaks due to a built-in cooling system. Nonetheless, heat can be dissipated into the samples during high-intensity milling, resulting in increased sample temperatures that potentially can affect thermolabile compounds and potential influence the obtained suspensions in the screening experiments if the used stabilizer has temperature dependent variations in the performance. Hence, a systematic investigation of the influence of different process parameters on the heat dissipation in samples during milling by the dual centrifugation approach was performed in the present study. It was found that the milling speed had the highest impact on the final sample temperature, but also other parameters, such as the bead loading, bead size, and placement in the centrifuge during milling had significantly influenced the final mean temperature of the milling media. Higher temperatures were obtained with higher bead loadings, i.e., 3000 mg milling beads/mL and milling speeds (1500 rpm), and when smaller milling beads, i.e., 0.1 mm, were used during production. The study further showed that higher temperatures were measured for samples located on the bottom disk during milling, and also when located on the outer placement on the sample disk. Upscale investigations showed immensely increased sample temperatures (almost up to boiling point) when samples were prepared under similar formulation parameters and milling speed as small-volume vials. Furthermore, the study indicated that the addition of drug compounds during suspension preparation decreased the final sample temperature compared to samples that only contained purified water due to energy absorption of the drug compound.

Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1's Behavior in Live Cells.

Uniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named "suspension bead loading" (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin-related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

Efficacy of CalliSpheres® drug-loaded microspheres combined with doxorubicin in hepatocellular carcinoma.

This study compared the efficacy and safety of the transarterial chemoembolization with CalliSpheres® drug-eluting beads loading with doxorubicin (DEB-TACE) versus conventional lipiodol (cTACE) in patients with unresectable hepatocellular carcinoma (HCC). A randomized controlled trial (RCT) was conducted with 144 patients, who were randomly assigned to receive either DEB-TACE with doxorubicin-loaded CalliSpheres® microspheres or cTACE with doxorubicin-lipiodol emulsion. Patients were followed up for 12 months, with assessments at 3 and 12 months posttreatment. The primary endpoint was the clinical response rate (CR), and the secondary endpoints were the overall survival (OS), the progression-free survival (PFS), and the safety profile of the two treatments. The results showed that DEB-TACE was superior to cTACE in terms of CR (50.0% vs 30.6% at 3 months, p = 0.03; 43.1% vs 25.0% at 12 months, p = 0.04), OS (18.2 months vs 14.6 months, p < 0.05), and PFS (7.4 months vs 4.8 months, p < 0.05), and that the safety profile of the two treatments was similar (p > 0.05 for all comparisons). However, the efficacy of DEB-TACE and cTACE varied according to the tumor morphology. DEB-TACE showed better CR rates in patients with nodular tumors, while no significant difference in CR between the two groups in patients with infiltrative tumors. DEB-TACE showed superior efficacy to cTACE in terms of CR, OS, and PFS, particularly in patients with nodular tumors, while maintaining a similar safety profile. These findings suggest that tumor morphology could inform treatment decisions for TACE in HCC patients.

Exploring the relationship between bulk Young’s Modulus of materials and milling efficiency during wet bead milling of pharmaceutical compounds

Wet bead milling (WBM) is one of the main approaches for manufacturing long acting injectable (LAI) suspensions, wherein the particle size of an Active Pharmaceutical Ingredient (API) is reduced in a liquid vehicle via grinding. A common challenge observed during WBM is long milling time to achieve target particle size, resulting in poor milling efficiency. The objective of this work was to identify potential API attributes predictive of milling efficiency during WBM. In this study, physical and mechanical properties of nine APIs were characterized. Formulations with these APIs were manufactured using WBM. Bulk Young’s Modulus was identified to have a significant influence on the rate of particle attrition. The rank order of Young’s Moduli of the APIs was consistent with that of milling efficiency, estimated by an empirical function defined in this study called Milling Resistance (ϕ), representing the holistic impact of milling time, tip speed, bead loading, and batch to chamber volume ratio. The identification of such intrinsic material properties, which provide an early evaluation of potential manufacturing risks, is beneficial to product development, as these assessments can be performed with limited quantities of materials and help identify and design out scale-up challenges.

Development of a Semi-Mechanistic Modeling Framework for Wet Bead Milling of Pharmaceutical Nanosuspensions.

This study aimed to develop a practical semi-mechanistic modeling framework to predict particle size evolution during wet bead milling of pharmaceutical nanosuspensions over a wide range of process conditions and milling scales. The model incorporates process parameters, formulation parameters, and equipment-specific parameters such as rotor speed, bead type, bead size, bead loading, active pharmaceutical ingredient (API) mass, temperature, API loading, maximum bead volume, blade diameter, distance between blade and wall, and an efficiency parameter. The characteristic particle size quantiles, i.e., x10, x50, and x90, were transformed to obtain a linear relationship with time, while the general functional form of the apparent breakage rate constant of this relationship was derived based on three models with different complexity levels. Model A, the most complex and general model, was derived directly from microhydrodynamics. Model B is a simpler model based on a power-law function of process parameters. Model C is the simplest model, which is the pre-calibrated version of Model B based on data collected from different mills across scales, formulations, and drug products. Being simple and computationally convenient, Model C is expected to reduce the amount of experimentation needed to develop and optimize the wet bead milling process and streamline scale-up and/or scale-out.

Influence of process parameters on bead geometry and its effect on mechanical properties of CMT welding of automotive grade steel and aluminium alloy using aluminium based filler wire

This investigation aims to examine the effectiveness of torch position, welding-brazing parameters on the embedded bead profile and load carrying potential of the brazed joint of zinc coated DP600 steel and Al 5052 joined by cold metal transfer (CMT) technique. The base metals were joined at different torch position (−0.6, 0 and 0.6 mm) in lap joint position using Al-based filler wire. At different wire feed rate (WFR) 3, 4 and 5 m/min changes in bead shape and its geometry were observed. The various modes of failure were classified based on effective bonding length (LH–LV) and LH/θ ratio. The best combination of welding-brazing parameters at which the maximum load bearing capacity of 4.1 kN and enhanced bead properties was achieved at 0.6 mm torch position and WFR 5 m/min. The intermetallic compound layers formed during the process affect the tensile properties of weld-brazed joint. The possible intermetallics and the presence of major elements in intermetallic layer were analyzed by energy-dispersive X-ray spectroscopic analysis. Furthermore, in this investigation, it was also observed that the thickness of IMC layer increases with the increasing WFR and heat input up to a critical value of 10 µm.

Do Mixtures of Beads with Different Sizes Improve Wet Stirred Media Milling of Drug Suspensions?

The impacts of bead sizes and bead mixtures on breakage kinetics, the number of milling cycles applied to prevent overheating, and power consumption during the nanomilling of drug (griseofulvin) suspensions were investigated from both an experimental and theoretical perspective. Narrowly sized zirconia beads with nominal sizes of 100, 200, and 400 µm and their half-and-half binary mixtures were used at 3000 and 4000 rpm with two bead loadings of 0.35 and 0.50. Particle size evolution was measured during the 3 h milling experiments using laser diffraction. An nth-order breakage model was fitted to the experimental median particle size evolution, and various microhydrodynamic parameters were calculated. In general, the beads and their mixtures with smaller median sizes achieved faster breakage. While the microhydrodynamic model explained the impacts of process parameters, it was limited in describing bead mixtures. For additional test runs performed, the kinetics model augmented with a decision tree model using process parameters outperformed that augmented with an elastic-net regression model using the microhydrodynamic parameters. The evaluation of the process merit scores suggests that the use of bead mixtures did not lead to notable process improvement; 100 µm beads generally outperformed bead mixtures and coarser beads in terms of fast breakage, low power consumption and heat generation, and low intermittent milling cycles.

A novel chitosan-coated Fe–Cu CNS loaded with CMC–Alginate composite for adsorptive removal of ciprofloxacin from water

Indiscriminate usage of antibiotic, Ciprofloxacin (CIPRO) poses a serious environmental threat by accumulating in the water bodies. A novel clay-nanosheet supported with Fe-Cu nanocomposite was developed for CIPRO removal using an eco-friendly approach. This composite was immobilised in a biodegradable chitosan-coated Alginate-CMC matrix to form nanocomposite beads for ease of usage in the column reactors. About 90% CIPRO removal was attained through batch mode under optimal conditions (20 mg/L CIPRO; bead loading 2000 mg/L; bead weight 0.15 g; contact period 300 min; and pH - 6). Effect of various environmental factors such as salts NaCl and CaCl2 (0.05, 0.1, 0.2, 0.5 and 1 M), and micro contaminants humic acid and polyethylene (1, 2, 3, 4 and 5 M) on CIPRO adsorption by NC beads was studied. Pseudo-second order kinetics was observed by the removal process and Langmuir isotherm by the adsorption with a maximum removal capacity of 485.58 mg/g. The effects of various reaction parameters such as the flow rate (1, 2 and 3 mL/min), initial CIPRO concentration (20, 40 and 60 mg/L) and bed height (3, 5 and 7 cm) were studied in the column reactors. The residual toxicity of composite beads interacted CIPRO solution was analysed with environmentally relevant algae such as Chlorella sp. and Scenedesmus obliquus, and the studies confirmed significant reduction in their toxic effects.

Polycaprolactone with Glass Beads for 3D Printing Filaments

At present, 3D printing is experiencing a great boom. The demand for new materials for 3D printing is also related to its expansion. This paper deals with manufacturing innovative polymer composite filaments suitable for the Fused Filament Fabrication method in 3D printing. As a filler, common and uncostly glass beads were used and mixed with biocompatible and biodegradable poly (ε-caprolactone), as a polymer matrix. This material was characterized via several physical-chemical methods. The Youngs modulus was increasing by about 30% with 20% loading of glass beads, and simultaneously, brittleness and elongations were decreased. The glass beads do not affect the shore hardness of filaments. The rheological measurement confirmed the material stability in a range of temperatures 75–120 °C. The presented work aimed to prepare lightweight biocompatible, cheap material with appropriate mechanical properties, lower printing temperature, and good printing processing. We can assess that the goal was fully met, and these filaments could be used for a wide range of applications.

Structure and rheology of oil-continuous capillary suspensions containing water-swellable cellulose beads and fibres

Cellulose particles are used in a range of materials and applications. Here, we investigated the role of cellulose sorptivity on the rheology and microstructure of oil-continuous capillary suspensions consisting of cellulose beads and fibres at volume fractions of 0.3–0.65 and water at volume fractions up to 0.2. We hypothesised that, in spite of their water-absorbent nature, cellulose particles in oil would be structured into a percolated network by water added as an immiscible secondary phase. The interplay between cellulose bead load and water content was used to generate suspensions ranging from flowable to highly elastic with moduli near 1 MPa. While capillary bridging induced a liquid to semi-solid transition, nearer particle volume fractions of 0.65, we found a transition to a solid, wet-granular material. Liquid-solid transitions occurred even if the water was absorbed by the particles, suggesting that the increase in elasticity was not solely due to the formation of capillary bridges, but also as a result of clustering and the associated increase in effective packing volume fraction. Swapping the beads with fibres increased stiffness by 4 orders whereas swapping half of the beads at the same water fraction increased material stiffness by 3 orders of magnitude. The two conclusions of this study were that, even for water-sorptive particles, capillary and hydrophilic interactions may be used to structure oil-continuous suspensions, and that the use of high aspect ratio particles can increase the rigidity of oil-continuous capillary suspensions to a greater extent than beads at an equivalent volume fraction.

Elaborating the crystal transformation referenced microhydrodynamic model and fracture mechanism combined molecular modelling of irbesartan nanosuspensions formation in wet media milling

In recent years, polymorphic transformation involved in media milling has become a key factor in inducing the instability of nanosuspensions (NSs). The variation trend of microhydrodynamic parameters, including milling intensity factor (F), can be observed under different milling conditions. Therefore, this study first referenced the microhydrodynamic model to explore how formulations and process parameters affect Irbesartan (IRB) form A crystallinity during wet media milling. As a result, the crystallinity of form A was affected by the intermolecular interactions between drug particles and stabilizers. The crystallinity of form A decreased with decreasing drug loading, increasing stirrer speed and bead loading, which depended on the role of F. Milling could promote the transformation from a 1H to 2H tetrazole ring with stabilizers containing –OH, and form B was changed to form A and finally to an amorphous state. Molecular modelling shows that forms A and B are ductile and fragile materials, respectively, and both present anisotropy. When milling beads hit both polymorphs paralleling to the (010) surface, the bead-bead collisions are more helpful in fracturing IRB particles. The results of this study may provide a foundation for controlling crystal transformation and obtaining ideal crystal forms.

Predicting the Temperature Evolution during Nanomilling of Drug Suspensions via a Semi-Theoretical Lumped-Parameter Model.

Although temperature can significantly affect the stability and degradation of drug nanosuspensions, temperature evolution during the production of drug nanoparticles via wet stirred media milling, also known as nanomilling, has not been studied extensively. This study aims to establish both descriptive and predictive capabilities of a semi-theoretical lumped parameter model (LPM) for temperature evolution. In the experiments, the mill was operated at various stirrer speeds, bead loadings, and bead sizes, while the temperature evolution at the mill outlet was recorded. The LPM was formulated and fitted to the experimental temperature profiles in the training runs, and its parameters, i.e., the apparent heat generation rate Qgen and the apparent overall heat transfer coefficient times surface area UA, were estimated. For the test runs, these parameters were predicted as a function of the process parameters via a power law (PL) model and machine learning (ML) model. The LPM augmented with the PL and ML models was used to predict the temperature evolution in the test runs. The LPM predictions were also compared with those of an enthalpy balance model (EBM) developed recently. The LPM had a fitting capability with a root-mean-squared error (RMSE) lower than 0.9 °C, and a prediction capability, when augmented with the PL and ML models, with an RMSE lower than 4.1 and 2.1 °C, respectively. Overall, the LPM augmented with the PL model had both good descriptive and predictive capability, whereas the one with the ML model had a comparable predictive capability. Despite being simple, with two parameters and obviating the need for sophisticated numerical techniques for its solution, the semi-theoretical LPM generally predicts the temperature evolution similarly or slightly better than the EBM. Hence, this study has provided a validated, simple model for pharmaceutical engineers to simulate the temperature evolution during the nanomilling process, which will help to set proper process controls for thermally labile drugs.

Visualizing Active Replication Regions in S-Phase Chromosomes.

A basic question of cell biology is how DNA folds to chromosome. A number of recently accumulated evidences have suggested that folding of chromosome proceeds tightly coupled with DNA replication progresses. Drug-induced PCC is a useful tool for visualization of the interphase nuclei, in particular, S-phase, as S-phase prematurely condensed chromosomes (S-phase PCC). Active replicating DNA is labeled directly with Cy3-dUTP by bead loading method, and then S-phase nuclei is immediately condensed prematurely by calyculin A to obtain S-phase PCC. Active replicating regions on S-PCC are observed under a scanning confocal microscope. Cy3-dUTP-labeled S-phase PCCs clearly reveal the drastic transitional change of chromosome formation through S-phase, starting from a "cloudy nebula" to numerous numbers of "beads on a string" and finally to "striped arrays of banding structured chromosome" known as G- or R-banding pattern. The number, distribution, and shape of replication foci were also measured in individual subphase of S-phase; maximally ~1400 foci of 0.35 μm average radius size were scored at the beginning of S-phase, and the number is reduced to ~100 at the end of S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for construction of eukaryote higher-ordered chromosome structure.

An Enthalpy-Balance Model for Timewise Evolution of Temperature during Wet Stirred Media Milling of Drug Suspensions.

Nanosuspensions have been used for enhancing the bioavailability of poorly soluble drugs. This study explores the temperature evolution during their preparation in a wet stirred media mill using a coupled experimental-enthalpy balance approach. Milling was performed at three levels of stirrer speed, bead loading, and bead sizes. Temperatures were recorded over time, then simulated using an enthalpy balance model by fitting the fraction of power converted to heat ξ. Moreover, initial and final power, ξ, and temperature profiles at 5 different test runs were predicted by power-law (PL) and machine learning (ML) approaches. Heat generation was higher at the higher stirrer speed and bead loading/size, which was explained by the higher power consumption. Despite its simplicity with a single fitting parameter ξ, the enthalpy balance model fitted the temperature evolution well with root mean squared error (RMSE) of 0.40-2.34°C. PL and ML approaches provided decent predictions of the temperature profiles in the test runs, with RMSE of 0.93-4.17 and 1.00-2.17°C, respectively. We established the impact of milling parameters on heat generation-power and demonstrated the simulation-prediction capability of an enthalpy balance model when coupled to the PL-ML approaches.

Floating Hydrogel Beads Made by Droplet Impact.

Droplet impact is a ubiquitous natural phenomenon that has been widely utilized to inspire and facilitate many industrial applications. Compared to the widely studied water droplet impact onto identical liquid surfaces, the water droplet impact onto an oil layer floating on a water bath (OLW) receives far less attention and its potential application has never been exploited. Herein, the process of water droplet impact onto the OLW is investigated with emphasis on the metastable states and potential applications. It is found that the dramatic deformation of the oil-water interface caused by the water droplet impact leads to two metastable states: oil in water in oil in water (O/W/O/W) and oil in water in oil (O/W/O). Through the subsequent introduction of gelation process, the metastable states can be frozen into floating hydrogel beads with similar shape to the roly-poly toys, which are attempted in gastric retentive drug delivery and algae bloom control. Specifically, the floating hydrogel beads perform well in gastric retentive drug delivery in vitro due to their inherent slow-release properties and floating capability. In addition, the floating hydrogel beads loading photocatalysts can capture more sunshine, and exhibit high photocatalytic efficiency, which is thus responsible for efficient algae bloom control.

Reduction of 4-nitrophenol using copper loaded surfactant-modified chitosan beads: An approach towards sludge management

Chitosan hydrogel (CS) beads and surfactant-modified chitosan hydrogel (SMCS) beads are used for the removal of Cu(II) ions from aqueous solution. After removal of metal ions, the waste Cu(II) ion-adsorbed beads have been used as an efficient catalyst for the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) in aqueous NaBH4. This approach can be considered as a part of ‘zero waste management practice’. The first step of the present work deals with the preparation of CS hydrogel beads with their further modification using anionic surfactant e.g. sodium dodecyl sulfate (SDS) at a concentration greater than the critical micelle concentration (CMC). This step leads to the formation of SMCS beads. In the second step, the CS and SMCS beads are used to remove Cu(II) ions from aqueous solution. The Cu(II) ion-loaded CS beads are designated as Cu-CS beads, and the Cu(II) ion-loaded SMCS beads are named as Cu-SMCS. These beads can be recognized as waste (or spent) beads. The Cu(II) ion removal efficiency and the calculated Cu(II) loading of SMCS beads are very similar to that of normal CS beads. In the third step these waste Cu-CS and Cu-SMCS beads have been utilized as a catalyst to reduce 4-NP to 4-AP in presence of borohydride. The 4-NP reduction using Cu-CS and Cu-SMCS follows zero order kinetics at varying catalyst doses in the range of 0.2–0.6 g/L, and at varying 4-NP concentrations in the range of 4 × 10−5 – 1.2 × 10−4 M. However, at all the doses the induction time required to start the reduction is higher in case of Cu-CS than Cu-SMCS beads. It is interesting to observe that, at a catalyst dose of 0.2 g/L and under identical condition, the Cu-SMCS beads act as a much better catalyst compared to the Cu-CS, and the zero order rate constant obtained with Cu-SMCS as the catalyst is found to be ~ 30 times higher compared to that obtained using Cu-CS. This is due to the synergistic effect offered by the surfactant bilayer present on the surface of Cu-SMCS. The characterization of the Cu-SMCS beads has been done using XRD, SEM, EDAX, FTIR analyses. The turnover number (TON) and turnover frequency (TOF) of Cu-SMCS are compared with other reported catalysts, and they are found comparable. Thus waste Cu-SMCS beads have been proved to be an effective catalyst for 4-NP reduction.

Analysis of heat generation during the production of drug nanosuspensions in a wet stirred media mill

Although heat is generated during the wet stirred media milling of drug suspensions, leading to notable temperature rise, a comprehensive analysis of heat generation does not exist. Hence, we investigated the impact of stirrer speed, bead loading, and bead size at three levels on the evolution of suspension temperature at the mill outlet during the milling of fenofibrate. The particle sizes and viscosities of the milled suspensions and power were measured. Our results suggest that stirrer speed had the most significant impact on the temperature increase, followed by bead loading and bead size. Both the time when the temperature reached 22 °C and the temperature at 5 min of milling were strongly correlated with the power. Assessing the impacts of the process parameters on the temperature rise, cycle time, power, and median particle size holistically, an optimal milling process was identified: 3000 rpm with 50% loading of 200 or 400 µm beads. A power number correlation was established to calculate power at any milling condition which determines the heat generation rate. Overall, this study indicated the importance of developing a good understanding of heat generation during nanomilling for development of a robust milling process especially for thermally labile drugs.

Synthetic Proteins behind the Plasma Barrier: Molecular Spies.

ConspectusThere is a continuous demand to improve our understanding of fundamental processes that underlie human health and disease. Therefore, novel strategies that can assist in these efforts are required. For example, molecular biology and genetic approaches have revolutionized our understanding of protein-mediated processes by facilitating their direct visualization and analyses in living cells. Despite these developments, genetic manipulation has limitations in controlling events that occur after translation such as posttranslational modifications (PTMs), which are imperative regulatory elements. As a result, developing new methods to study PTMs in live cells is a major bottleneck in deciphering their exact roles in the myriad cellular processes.Synthetic and semisynthetic proteins are prepared by combining solid phase peptide synthesis (SPPS) and chemoselective ligation approaches with synthetic or recombinant peptides. Employing protein synthesis allows chemists to incorporate natural and unnatural modifications with virtually unlimited number of functional groups into the protein’s sequence, such as PTMs and their mimics. In addition, synthetic proteins can include additional elements such as fluorescent tags, reactive groups, caged units, and enrichment handles. Therefore, harnessing the power of chemical protein synthesis offers great opportunities to study fundamental biological processes.Unfortunately, the low cell permeability of proteins limits their applications mainly to in vitro settings, excluding live cell studies. As a result, chemical biologists have been attempting to overcome these limitations by developing protein delivery methods that would enable the study of custom-made proteins in a biological context. Success with these strategies should enable accurate determination of protein localization, degradation, folding, interactions, and involvement in the assembly of membrane-less organelles formed by liquid–liquid phase separation inside cells. Importantly, protein delivery approaches are complementary to genetic manipulations, and combining these approaches should pave the way to new discoveries.In this Account, we describe recent developments in protein delivery methods, with emphasis on those most compatible with synthetic proteins. We highlight experimental approaches and conceptual adaptations required to design and study synthetic proteins in live cells, with or without genetic manipulation. In addition, we highlight the strength and weakness of these approaches for both the delivery and the subsequent studies. We also describe our endeavors to deliver synthetic proteins to cells via cell penetrating peptides (CPPs) and multiplexed bead loading (MBL), as showcases of the applications of these methods to shed light on biological processes. Lastly, we contemplate other future applications of synthetic proteins to answer questions that are currently unapproachable.