The Impact of Protein Corona Formation on the Macrophage Cellular Uptake and Biodistribution of Spherical Nucleic Acids

The effect of serum protein adsorption on the biological fate of Spherical Nucleic Acids (SNAs) is investigated. Through a proteomic analysis, it is shown that G‐quadruplexes templated on the surface of a gold nanoparticle in the form of SNAs mediate the formation of a protein corona that is rich in complement proteins relative to SNAs composed of poly‐thymine (poly‐T) DNA. Cellular uptake studies show that complement receptors on macrophage cells recognize the SNA protein corona, facilitating their internalization, and causing G‐rich SNAs to accumulate in the liver and spleen more than poly‐T SNAs in vivo. These results support the conclusion that nucleic acid sequence and architecture can mediate nanoparticle–biomolecule interactions and alter their cellular uptake and biodistribution properties and illustrate that nucleic acid sequence is an important parameter in the design of SNA therapeutics.     

Stressing the Role of DNA as a Drug Carrier: Synthesis of DNA–Drug Conjugates through Grafting Chemotherapeutics onto Phosphorothioate Oligonucleotides

To stress the role of deoxyribonucleic acid (DNA) as a drug carrier, an efficient conjugation strategy in which chemotherapeutics can be grafted onto a phosphorothiolated DNA backbone through the reaction between the phosphorothioate group (PS) and a benzyl bromide group is proposed. As a proof of concept, benzyl‐bromide‐modified paclitaxel (PTX) is employed to graft onto the DNA backbone at the PS modification sites. Due to the easy preparation of phosphorothiolated DNA at any desired position during its solid‐phase synthesis, diblock DNA strands containing both normal phosphodiester segment (^PODNA) and phosphorothiolate segment (^PSDNA) are directly grafted with a multitude of PTXs without using complicated and exogenous linkers. Then, the resulting amphiphilic ^PODNA‐blocked‐(^PSDNA‐grafted PTX) conjugates (^PODNA‐b‐(^PSDNA‐g‐PTX)) assemble into PTX‐loaded spherical nucleic acid (SNA)‐like micellar nanoparticles (PTX‐SNAs) with a high drug loading ratio up to ≈53%. Importantly, the ^PODNA segment maintains its molecular recognition property and biological functions, which allows the as‐prepared PTX‐SNAs to be further functionalized with tumor‐targeting aptamers, fluorescent probe strands, or antisense sequences. These multifunctional PTX‐SNAs demonstrate active tumor‐targeting delivery, efficient inhibition of tumor growth, and the reversal of drug resistance both in vitro and in vivo for comprehensive antitumor therapy.     

The Sequence‐Specific Cellular Uptake of Spherical Nucleic Acid Nanoparticle Conjugates

The sequence‐dependent cellular uptake of spherical nucleic acid nanoparticle conjugates (SNAs) is investigated. This process occurs by interaction with class A scavenger receptors (SR‐A) and caveolae‐mediated endocytosis. It is known that linear poly(guanine) (poly G) is a natural ligand for SR‐A, and it has been proposed that interaction of poly G with SR‐A is dependent on the formation of G‐quadruplexes. Since G‐rich oligonucleotides are known to interact strongly with SR‐A, it is hypothesized that SNAs with higher G contents would be able to enter cells in larger amounts than SNAs composed of other nucleotides, and as such, cellular internalization of SNAs is measured as a function of constituent oligonucleotide sequence. Indeed, SNAs with enriched G content show the highest cellular uptake. Using this hypothesis, a small molecule (camptothecin) is chemically conjugated with SNAs to create drug‐SNA conjugates and it is observed that poly G SNAs deliver the most camptothecin to cells and have the highest cytotoxicity in cancer cells. Our data elucidate important design considerations for enhancing the intracellular delivery of spherical nucleic acids.     

Exosome Encased Spherical Nucleic Acid Gold Nanoparticle Conjugates as Potent MicroRNA Regulation Agents

Exosomes are a class of naturally occurring nanomaterials that play crucial roles in the protection and transport of endogenous macromolecules, such as microRNA and mRNA, over long distances. Intense effort is underway to exploit the use of exosomes to deliver synthetic therapeutics. Herein, transmission electron microscopy is used to show that when spherical nucleic acid (SNA) constructs are endocytosed into PC‐3 prostate cancer cells, a small fraction of them (<1%) can be naturally sorted into exosomes. The exosome‐encased SNAs are secreted into the extracellular environment from which they can be isolated and selectively re‐introduced into the cell type from which they were derived. In the context of anti‐miR21 experiments, the exosome‐encased SNAs knockdown miR‐21 target by approximately 50%. Similar knockdown of miR‐21 by free SNAs requires a ≈3000‐fold higher concentration.     

Synthesis and Assembly of Click‐Nucleic‐Acid‐Containing PEG–PLGA Nanoparticles for DNA Delivery

Co‐delivery of both chemotherapy drugs and siRNA from a single delivery vehicle can have a significant impact on cancer therapy due to the potential for overcoming issues such as drug resistance. However, the inherent chemical differences between charged nucleic acids and hydrophobic drugs have hindered entrapment of both components within a single carrier. While poly(ethylene glycol)‐block‐poly(lactic‐co‐glycolic acid) (PEG–PLGA) copolymers have been used successfully for targeted delivery of chemotherapy drugs, loading of DNA or RNA has been poor. It is demonstrated that significant amounts of DNA can be encapsulated within PLGA‐containing nanoparticles through the use of a new synthetic DNA analog, click nucleic acids (CNAs). First, triblock copolymers of PEG‐CNA‐PLGA are synthesized and then formulated into polymer nanoparticles from oil‐in‐water emulsions. The CNA‐containing particles show high encapsulation of DNA complementary to the CNA sequence, whereas PEG‐PLGA alone shows minimal DNA loading, and non‐complementary DNA strands do not get encapsulated within the PEG‐CNA‐PLGA nanoparticles. Furthermore, the dye pyrene can be successfully co‐loaded with DNA and lastly, a complex, larger DNA sequence that contains an overhang complementary to the CNA can also be encapsulated, demonstrating the potential utility of the CNA‐containing particles as carriers for chemotherapy agents and gene silencers.     

Inhibition of 3‐D Tumor Spheroids by Timed‐Released Hydrophilic and Hydrophobic Drugs from Multilayered Polymeric Microparticles

First‐line cancer chemotherapy necessitates high parenteral dosage and repeated dosing of a combination of drugs over a prolonged period. Current commercially available chemotherapeutic agents, such as Doxil and Taxol, are only capable of delivering single drug in a bolus dose. The aim of this study is to develop dual‐drug‐loaded, multilayered microparticles and to investigate their antitumor efficacy compared with single‐drug‐loaded particles. Results show hydrophilic doxorubicin HCl (DOX) and hydrophobic paclitaxel (PTX) localized in the poly(dl ‐lactic‐co‐glycolic acid, 50:50) (PLGA) shell and in the poly(l ‐lactic acid) (PLLA) core, respectively. The introduction of poly[(1,6‐bis‐carboxyphenoxy) hexane] (PCPH) into PLGA/PLLA microparticles causes PTX to be localized in the PLLA and PCPH mid‐layers, whereas DOX is found in both the PLGA shell and core. PLGA/PLLA/PCPH microparticles with denser shells allow better control of DOX release. A delayed release of PTX is observed with the addition of PCPH. Three‐dimensional MCF‐7 spheroid studies demonstrate that controlled co‐delivery of DOX and PTX from multilayered microparticles produces a greater reduction in spheroid growth rate compared with single‐drug‐loaded particles. This study provides mechanistic insights into how distinctive structure of multilayered microparticles can be designed to modulate the release profiles of anticancer drugs, and how co‐delivery can potentially provide better antitumor response.     

Hollow spherical nucleic acids for intracellular gene regulation based upon biocompatible silica shells

Cellular transfection of nucleic acids is necessary for regulating gene expression through antisense or RNAi pathways. The development of spherical nucleic acids (SNAs, originally gold nanoparticles functionalized with synthetic oligonucleotides) has resulted in a powerful set of constructs that are able to efficiently transfect cells and regulate gene expression without the use of auxiliary cationic cocarriers. The gold core in such structures is primarily used as a template to arrange the nucleic acids into a densely packed and highly oriented form. In this work, we have developed methodology for coating the gold particle with a shell of silica, modifying the silica with a layer of oligonucleotides, and subsequently oxidatively dissolving the gold core with I(2). The resulting hollow silica-based SNAs exhibit cooperative binding behavior with respect to complementary oligonucleotides and cellular uptake properties comparable to their gold-core SNA counterparts. Importantly, they exhibit no cytotoxicity and have been used to effectively silence the eGFP gene in mouse endothelial cells through an antisense approach.     

One‐Step Microfluidic Synthesis of Nanocomplex with Tunable Rigidity and Acid‐Switchable Surface Charge for Overcoming Drug Resistance

Multidrug resistance (MDR), is the key reason accounting for the failure of cancer chemotherapy, remains a dramatic challenge for cancer therapy. In this study, the one‐step microfluidic fabrication of a rigid pH‐sensitive micellar nanocomplex (RPN) with tunable rigidity and acid‐switchable surface charge for overcoming MDR by enhancing cellular uptake and lysosome escape is demonstrated. The RPN is composed of a poly(lactic‐co‐glycolic acid) (PLGA) core and a pH‐sensitive copolymer shell, which is of neutral surface charge during blood circulation. Upon internalization of RPN by cancer cells, the pH‐responsive shell dissociates inside the acidic lysosomes, while the rigid and positively charged PLGA core improves the lysosomal escape. The cellular uptake and nuclear uptake of doxorubicin (Dox) from Dox‐loaded RPN are 1.6 and 2.4 times higher than that from Dox‐loaded pH‐sensitive micelles (PM) using a Dox‐resistant cancer model (MCF‐7/ADR, re‐designated NCI/ADR‐RES) in vitro. Dox‐loaded RPN significantly enhances the therapeutic efficacy (92% inhibition of tumor growth) against MCF‐7/ADR xenograft tumor in mice, while Dox‐loaded PM only inhibits the tumor growth by 36%. RPN avoids the use of complicated synthesis procedure of nanoparticle and the necessary to integrate multiple components, which can facilitate the clinical translation of this novel nanostructure.     

Dual Drug Backboned Shattering Polymeric Theranostic Nanomedicine for Synergistic Eradication of Patient‐Derived Lung Cancer

Most of the current nanoparticle‐based therapeutics worldwide failing in clinical trials face three major challenges: (i) lack of an optimum drug delivery platform with precise composition, (ii) lack of a method of directly monitoring the fate of a specific drug rather than using any other labelling molecules as a compromise, and (iii) lack of reliable cancer models with high fidelity for drug screen and evaluation. Here, starting from a PP2A inhibitor demethylcantharidin (DMC) and cisplatin, the design of a dual sensitive dual drug backboned shattering polymer (DDBSP) with exact composition at a fixed DMC/Pt ratio for precise nanomedicine is shown. DDBSP self‐assembled nanoparticle (DD‐NP) can be triggered intracellularly to break down in a chain‐shattering manner to release the dual drugs payload. Moreover, DD‐NP with extremely high Pt heavy metal content in the polymer chain can directly track the drug itself via Pt‐based drug‐mediated computer tomography and ICP‐MS both in vitro and in vivo. Finally, DD‐NP is used to eradicate the tumor burden on a high‐fidelity patient‐derived lung cancer model for the first time.     

Low Molecular Weight Heparin-Loaded Polymeric Nanoparticles: Formulation,Characterization, and Release Characteristics

ABSTRACT

The aim of the present work was to investigate the preparation of low molecular weight heparin (LMWH) nanoparticles (NP) as potential oral heparin carriers. The NP were formulated using an ultrasound probe by water-in-oil-in-water (w/o/w) emulsification and solvent evaporation with two biodegradable polymers [poly-ε-caprolactone, PCL and poly(d,l-lactic-co-glycolic acid) 50/50, PLGA] and two non-biodegradable positively charged polymers (Eudragit RS and RL) used alone or in combination. The mean diameter of LMWH-loaded NP ranged from 240 to 490 nm and was dependent on the reduced viscosity of the polymeric organic solution. The surface potential of LMWH NP prepared with Eudragit polymers used alone or blended with PCL and PLGA was changed dramatically from strong positive values obtained with unloaded NP to negative values. The highest encapsulation efficiencies were observed when Eudragit polymers took part in the composition of the polymeric matrix, compared with PCL and PLGA NP exhibiting low LMWH entrapment. The in vitro LMWH release in phosphate buffer from all formulations ranged from 10 to 25% and was more important (two- to threefold) when esterase was added into the dissolution medium. The in vitro biological activity of released LMWH, determined by the anti-factor Xa activity with a chromogenic substrate, was preserved after the encapsulation process, making these NP good candidates for oral administration.     

Detection of β‐Amyloid by Sialic Acid Coated Bovine Serum Albumin Magnetic Nanoparticles in a Mouse Model of Alzheimer's Disease

The accumulation and formation of β‐amyloid (Aβ) plaques in the brain are distinctive pathological hallmarks of Alzheimer's disease (AD). Designing nanoparticle (NP) contrast agents capable of binding with Aβ highly selectively can potentially facilitate early detection of AD. However, a significant obstacle is the blood brain barrier (BBB), which can preclude the entrance of NPs into the brain for Aβ binding. In this work, bovine serum albumin (BSA) coated NPs are decorated with sialic acid (NP‐BSA_x‐Sia) to overcome the challenges in Aβ imaging in vivo. The NP‐BSA_x‐Sia is biocompatible with high magnetic relaxivities, suggesting that they are suitable contrast agents for magnetic resonance imaging (MRI). The NP‐BSA_x‐Sia binds with Aβ in a sialic acid dependent manner with high selectivities toward Aβ deposited on brains and cross the BBB in an in vitro model. The abilities of these NPs to detect Aβ in vivo in human AD transgenic mice by MRI are evaluated without the need to coinject mannitol to increase BBB permeability. T₂*‐weighted MRI shows that Aβ plaques in mouse brains can be detected as aided by NP‐BSA_x‐Sia, which is confirmed by histological analysis. Thus, NP‐BSA_x‐Sia is a promising new tool for noninvasive in vivo detection of Aβ plaques.     

In Vivo Behavior of Ultrasmall Spherical Nucleic Acids

The biological properties of spherical nucleic acids (SNAs) are largely independent of nanoparticle core identity but significantly affected by oligonucleotide surface density. Additionally, the payload-to-carrier (i.e., DNA-to-nanoparticle) mass ratio of SNAs is inversely proportional to core size. While SNAs with many core types and sizes have been developed, all in vivo analyses of SNA behavior have been limited to cores >10 nm in diameter. However, “ultrasmall” nanoparticle constructs (<10 nm diameter) can exhibit increased payload-to-carrier ratios, reduced liver accumulation, renal clearance, and enhanced tumor infiltration. Therefore, we hypothesized that SNAs with ultrasmall cores exhibit SNA-like properties, but with in vivo behavior akin to traditional ultrasmall nanoparticles. To investigate, we compared the behavior of SNAs with 1.4-nm Au₁₀₂ nanocluster cores (AuNC-SNAs) and SNAs with 10-nm gold nanoparticle cores (AuNP-SNAs). Significantly, AuNC-SNAs possess SNA-like properties (e.g., high cellular uptake, low cytotoxicity) but show distinct in vivo behavior. When intravenously injected in mice, AuNC-SNAs display prolonged blood circulation, lower liver accumulation, and higher tumor accumulation than AuNP-SNAs. Thus, SNA-like properties persist at the sub-10-nm length scale and oligonucleotide arrangement and surface density are responsible for the biological properties of SNAs. This work has implications for the design of new nanocarriers for therapeutic applications.     

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T‐cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non‐Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose‐dependent and time‐controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery.     

Nanoparticle‐Enhanced Radiotherapy to Trigger Robust Cancer Immunotherapy

External radiotherapy is extensively used in clinic to destruct tumors by locally applied ionizing‐radiation beams. However, the efficacy of radiotherapy is usually limited by tumor hypoxia‐associated radiation resistance. Moreover, as a local treatment technique, radiotherapy can hardly control tumor metastases, the major cause of cancer death. Herein, core–shell nanoparticles based poly(lactic‐co‐glycolic) acid (PLGA) are fabricate, by encapsulating water‐soluble catalase (Cat), an enzyme that can decompose H₂O₂ to generate O₂, inside the inner core, and loading hydrophobic imiquimod (R837), a Toll‐like‐receptor‐7 agonist, within the PLGA shell. The formed PLGA‐R837@Cat nanoparticles can greatly enhance radiotherapy efficacy by relieving the tumor hypoxia and modulating the immune‐suppressive tumor microenvironment. The tumor‐associated antigens generated postradiotherapy‐induced immunogenic cell death in the presence of such R837‐loaded adjuvant nanoparticles will induce strong antitumor immune responses, which together with cytotoxic T‐lymphocyte associated protein 4 (CTLA‐4) checkpoint blockade will be able to effectively inhibit tumor metastases by a strong abscopal effect. Moreover, a long term immunological memory effect to protect mice from tumor rechallenging is observed post such treatment. This work thus presents a unique nanomedicine approach as a next‐generation radiotherapy strategy to enable synergistic whole‐body therapeutic responses after local treatment, greatly promising for clinical translation.     

Memantine‐Loaded PEGylated Biodegradable Nanoparticles for the Treatment of Glaucoma

Glaucoma is a multifactorial neurodegenerative disease associated with retinal ganglion cells (RGC) loss. Increasing reports of similarities in glaucoma and other neurodegenerative conditions have led to speculation that therapies for brain neurodegenerative disorders may also have potential as glaucoma therapies. Memantine is an N‐methyl‐d ‐aspartate (NMDA) antagonist approved for Alzheimer's disease treatment. Glutamate‐induced excitotoxicity is implicated in glaucoma and NMDA receptor antagonism is advocated as a potential strategy for RGC preservation. This study describes the development of a topical formulation of memantine‐loaded PLGA‐PEG nanoparticles (MEM‐NP) and investigates the efficacy of this formulation using a well‐established glaucoma model. MEM‐NPs <200 nm in diameter and incorporating 4 mg mL^?1 of memantine were prepared with 0.35 mg mL^?1 localized to the aqueous interior. In vitro assessment indicated sustained release from MEM‐NPs and ex vivo ocular permeation studies demonstrated enhanced delivery. MEM‐NPs were additionally found to be well tolerated in vitro (human retinoblastoma cells) and in vivo (Draize test). Finally, when applied topically in a rodent model of ocular hypertension for three weeks, MEM‐NP eye drops were found to significantly (p < 0.0001) reduce RGC loss. These results suggest that topical MEM‐NP is safe, well tolerated, and, most promisingly, neuroprotective in an experimental glaucoma model.     

PLGA–PEG–PLGA hydrogel for ocular drug delivery of dexamethasone acetate

Aim: This study aims to investigate the suitability of thermosensitive triblock polymer poly-(dl-lactic acid-co-glycolic acid) (PLGA)–polyethylene glycol (PEG)–PLGA as a matrix material for ocular delivery of dexamethasone acetate (DXA). Methods: The copolymer was synthesized and evaluated for its thermosensitive and gelation properties. DXA in situ gel-forming solution based on PLGA–PEG–PLGA copolymer of 20% (w/w) was prepared and evaluated for ocular pharmacokinetics in rabbit according to the microdialysis method, which was compared to the normal eye drop. Result: The copolymer with 20% (w/w) had a low critical solution temperature of 32°C, which is close to the surface temperature of the eye. The C_max of DXA in the anterior chamber for the PLGA–PEG–PLGA solution was 125.2 μg/mL, which is sevenfold higher than that of the eye drop, along with greater area under the concentration–time curves (AUC). Conclusion: These results suggest that the PLGA–PEG–PLGA copolymer is potential thermosensitive in situ gel-forming material for ocular drug delivery, and it may improve the bioavailability, efficacy of some eye drugs.     

Dual Growth Factor Delivery Using Biocompatible Core–Shell Microcapsules for Angiogenesis

An optimized electrodropping system produces homogeneous core–shell microcapsules (C‐S MCs) by using poly(L ‐lactic‐co‐glycolic acid) (PLGA) and alginate. Fluorescence imaging clearly shows the C‐S domain in the MC. For release control, the use of high‐molecular‐weight PLGA (HMW 270 000) restrains the initial burst release of protein compared to that of low‐MW PLGA (LMW 40 000). Layer‐by‐layer (LBL) assembly of chitosan and alginate on MCs is also useful in controlling the release profile of biomolecules. LBL (7‐layer) treatment is effective in suppressing the initial burst release of protein compared to no LBL (0‐layer). The difference of cumulative albumin release between HMW (7‐layer LBL) and LMW (0‐layer LBL) PLGA is determined to be more than 40% on day 5. When dual angiogenic growth factors (GFs), such as platelet‐derived GF (PDGF) and vascular endothelial GF (VEGF), are encapsulated separately in the core and shell domains, respectively, the VEGF release rate is much greater than that of PDGF, and the difference of the cumulative release percentage between the two GFs is about 30% on day 7 with LMW core PLGA and more than 45% with HMW core PLGA. As for the angiogenic potential of MC GFs with human umbilical vein endothelial cells (HUVECs), the fluorescence signal of CD31+ suggests that the angiogenic sprout of ECs is more active in MC‐mediated GF delivery than conventional GF delivery, and this difference is significant, based on the number of capillary branches in the unit area. This study demonstrates that the fabrication of biocompatible C‐S MCs is possible, and that the release control of biomolecules is adjustable. Furthermore, MC‐mediated GFs remain in an active form and can upregulate the angiogenic activity of ECs.     

SN-38 loading capacity of hydrophobic polymer blend nanoparticles: formulation,optimization and efficacy evaluation

One of the most important problems in nanoencapsulation of extremely hydrophobic drugs is poor drug loading due to rapid drug crystallization outside the polymer core. The effort to use nanoprecipitation, as a simple one-step procedure with good reproducibility and FDA approved polymers like Poly(lactic-co-glycolic acid) (PLGA) and Polycaprolactone (PCL), will only potentiate this issue. Considering that drug loading is one of the key defining characteristics, in this study we attempted to examine whether the nanoparticle (NP) core composed of two hydrophobic polymers will provide increased drug loading for 7-Ethyl-10-hydroxy-camptothecin (SN-38), relative to NPs prepared using individual polymers. D-optimal design was applied to optimize PLGA/PCL ratio in the polymer blend and the mode of addition of the amphiphilic copolymer Lutrol^®F127 in order to maximize SN-38 loading and obtain NPs with acceptable size for passive tumor targeting. Drug/polymer and polymer/polymer interaction analysis pointed to high degree of compatibility and miscibility among both hydrophobic polymers, providing core configuration with higher drug loading capacity. Toxicity studies outlined the biocompatibility of the blank NPs. Increased in vitro efficacy of drug-loaded NPs compared to the free drug was confirmed by growth inhibition studies using SW-480 cell line. Additionally, the optimized NP formulation showed very promising blood circulation profile with elimination half-time of 7.4?h.     

Influence of Temperature on the Colloidal Stability of Polymer‐Coated Gold Nanoparticles in Cell Culture Media

The temperature‐dependence of the hydrodynamic diameter and colloidal stability of gold‐polymer core‐shell particles with temperature‐sensitive (poly(N‐isopropylacrylamide)) and temperature‐insensitive shells (polyallylaminine hydrochloride/polystyrensulfonate, poly(isobutylene‐alt‐maleic anhydride)‐graft‐dodecyl) are investigated in various aqueous media. The data demonstrate that for all nanoparticle agglomeration, i.e., increase in effective nanoparticle size, the presence of salts or proteins in the dispersion media has to be taken into account. Poly(N‐isopropylacrylamide) coated nanoparticles show a reversible temperature‐dependent increase in size above the volume phase transition of the polymer shell when they are dispersed in phosphate buffered saline or in media containing protein. In contrast, the nanoparticles coated with temperature‐insensitive polymers show a time‐dependent increase in size in phosphate buffered saline or in medium containing protein. This is due to time‐dependent agglomeration, which is particularly strong in phosphate buffered saline, and induces a time‐dependent, irreversible increase in the hydrodynamic diameter of the nanoparticles. This demonstrates that one has to distinguish between temperature‐ and time‐induced agglomerations. Since the size of nanoparticles regulates their uptake by cells, temperature‐dependent uptake of thermosensitive and non‐thermosensitive nanoparticles by cells lines is compared. No temperature‐specific difference between both types of nanoparticles could be observed.