Injectable Anhydrous Poly(ethylene glycol) Polymer Liquids Form Protein Depot for Extended Controlled Release Applications Via Rapid In Situ Gelation.

Water-free preparation of protein delivery systems has the potential to overcome the limitations of hydrogel depot systems such as off-target reactions, functional group hydrolysis, and limited loading capacity. However, a major roadblock in the development and use of these systems is administration as implantation is often required. In this study, we developed a biodegradable and water-free injectable protein delivery system via inverse electron demand Diels-Alder reaction between norbornene- and tetrazine-functionalized four-armed poly(ethylene glycol) macromonomers. 1:1 mixtures of these precursors gelled rapidly in situ, taking less than 11 s to reach their gelation point. Methyl substitution of tetrazine slowed the gelation time and increased the cross-linking density, whereas oxygen incorporation into norbornene changed the mechanical properties. Introduction of hydrolytically cleavable groups enabled biodegradability. Using phenyl carbamate and phenyl carbonate ester groups, we could tune the stability. Controlled release of the protein surrogate glucose oxidase was achieved over a period of 500 days. The novel preparation method presented here is a promising step toward the development of water-free injectable protein depots for controlled drug delivery.

Enzyme-triggered antigen release enhances cross-presentation by dendritic cells.

Nanoparticles hold great potential as vaccine carriers due to their highly versatile structure and the possibility to influence intracellular trafficking and antigen presentation by their design. In this study, we developed a nanoparticulate system with a new enzyme-triggered antigen release mechanism. For this novel approach, nanoparticle and model antigen ovalbumin were linked with a substrate of the early endosomal protease cathepsin S. This construct enabled the transfer of antigens delivered to bone marrow-derived dendritic cells from the endo-lysosomal compartments in the cytosol. Consecutively, our particles enhanced cross-presentation on dendritic cells and subsequently promoted a stronger activation of CD8+ T cells. Our findings suggest that enzyme-triggered antigen release allows the endosomal escape of the antigen, leading to increased MHC-I presentation. Since T cell immunity is central for the control of viral infections and cancer, this release mechanism offers a promising approach for the development of both prophylactic and therapeutic vaccines.

Influenza A virus mimetic nanoparticles trigger selective cell uptake.

Poor target cell specificity is currently a major shortcoming of nanoparticles (NPs) used for biomedical applications. It causes significant material loss to off-target sites and poor availability at the intended delivery site. To overcome this limitation, we designed particles that identify cells in a virus-like manner. As a blueprint, we chose a mechanism typical of influenza A virus particles in which ectoenzymatic hemagglutinin activation by target cells is a mandatory prerequisite for binding to a secondary target structure that finally confirms cell identity and allows for uptake of the virus. We developed NPs that probe mesangial cells for the presence of angiotensin-converting enzyme on their surface using angiotensin I (Ang-I) as a proligand. This initial interaction enzymatically transforms Ang-I to a secondary ligand angiotensin II (Ang-II) that has the potential to bind in a second stage to Ang-II type-1 receptor (AT1R). The presence of the receptor confirms the target cell identity and triggers NP uptake via endocytosis. Our virus-mimetic NPs showed outstanding target-cell affinity with picomolar avidities and were able to selectively identify these cells in the presence of 90% off-target cells that carried only the AT1R. Our results demonstrate that the design of virus-mimetic cell interactive NPs is a valuable strategy to enhance NP specificity for therapeutic and diagnostic applications. Our set of primary and secondary targets is particularly suited for the identification of mesangial cells that play a pivotal role in diabetic nephropathy, one of the leading causes of renal failure, for which currently no treatment exists.

In Situ Forming iEDDA Hydrogels with Tunable Gelation Time Release High-Molecular Weight Proteins in a Controlled Manner over an Extended Time.

Off-target interactions between reactive hydrogel moieties and drug cargo as well as slow reaction kinetics and the absence of controlled protein release over an extended period of time are major drawbacks of chemically cross-linked hydrogels for biomedical applications. In this study, the inverse electron demand Diels-Alder (iEDDA) reaction between norbornene- and tetrazine-functionalized eight-armed poly(ethylene glycol) (PEG) macromonomers was used to overcome these obstacles. Oscillatory shear experiments revealed that the gel point of a 15% (w/v) eight-armed PEG hydrogel with a molecular weight of 10 kDa was less than 15 s, suggesting the potential for fast in situ gelation. However, the high-speed reaction kinetics result in a risk of premature gel formation that complicates the injection process. Therefore, we investigated the effect of polymer concentration, temperature, and chemical structure on the gelation time. The cross-linking reaction was further characterized regarding bioorthogonality. Only 11% of the model protein lysozyme was found to be PEGylated by the iEDDA reaction, whereas 51% interacted with the classical Diels-Alder reaction. After determination of the mesh size, fluorescein isothiocyanate-dextran was used to examine the release behavior of the hydrogels. When glucose oxidase was embedded into 15% (w/v) hydrogels, a controlled release over more than 250 days was achieved. Overall, the PEG-based hydrogels cross-linked via the fast iEDDA reaction represent a promising material for the long-term administration of biologics.

Targeted Delivery of Soluble Guanylate Cyclase (sGC) Activator Cinaciguat to Renal Mesangial Cells via Virus-Mimetic Nanoparticles Potentiates Anti-Fibrotic Effects by cGMP-Mediated Suppression of the TGF-ß Pathway.

Diabetic nephropathy (DN) ranks among the most detrimental long-term effects of diabetes, affecting more than 30% of all patients. Within the diseased kidney, intraglomerular mesangial cells play a key role in facilitating the pro-fibrotic turnover of extracellular matrix components and a progredient glomerular hyperproliferation. These pathological effects are in part caused by an impaired functionality of soluble guanylate cyclase (sGC) and a consequentially reduced synthesis of anti-fibrotic messenger 3',5'-cyclic guanosine monophosphate (cGMP). Bay 58-2667 (cinaciguat) is able to re-activate defective sGC; however, the drug suffers from poor bioavailability and its systemic administration is linked to adverse events such as severe hypotension, which can hamper the therapeutic effect. In this study, cinaciguat was therefore efficiently encapsulated into virus-mimetic nanoparticles (NPs) that are able to specifically target renal mesangial cells and therefore increase the intracellular drug accumulation. NP-assisted drug delivery thereby increased in vitro potency of cinaciguat-induced sGC stabilization and activation, as well as the related downstream signaling 4- to 5-fold. Additionally, administration of drug-loaded NPs provided a considerable suppression of the non-canonical transforming growth factor ß (TGF-ß) signaling pathway and the resulting pro-fibrotic remodeling by 50-100%, making the system a promising tool for a more refined therapy of DN and other related kidney pathologies.

Thermodynamic, Spatial and Methodological Considerations for the Manufacturing of Therapeutic Polymer Nanoparticles.

PURPOSE: Evaluate fundamental parameters that dictate the effectiveness of drug loading. METHODS: A model water-soluble drug lacking ionizable groups, pirfenidone (PFD), was encapsulated through nanoprecipitation in poly(ethylene glycol)-poly(lactic acid) (PEG-PLA)-poly(lactic-co-glycolic acid) (PLGA) NPs. Firstly, the thermodynamic parameters predicting drug-polymer miscibility were determined to assess the system's suitability. Then, the encapsulation was evaluated experimentally by two different techniques, bulk and microfluidic (MF) nanoprecipitation. Additionally, the number of molecules that fit in a particle core were calculated and the loading determined experimentally for different core sizes. Lastly, the effect of co-encapsulation of α-lipoic acid (LA), a drug with complementary therapeutic effects and enhanced lipophilicity, was evaluated. RESULTS: The thermodynamic miscibility parameters predicted a good suitability of the selected system. MF manufacturing enhanced the encapsulation efficiency by 60-90% and achieved a 2-fold higher NP cellular uptake. Considering spatial constrictions for drug encapsulation and increasing the size of the PLGA core the number of PFD molecules per NP was raised from under 500 to up to 2000. More so, the co-encapsulation of LA increased the number of drug molecules per particle by 96%, with no interference with the release profile. CONCLUSIONS: Thermodynamic, spatial and methodological parameters should be considered to optimize drug encapsulation.

Fluorescent Nanoparticles Coated with a Somatostatin Analogue Target Blood Monocyte for Efficient Leukaemia Treatment.

BACKGROUND: Leukaemia is the most prevalent form of cancer-causing death in a large number of populations and needs prompt and effective treatment. Chemotherapeutics can be used to treat leukaemia, but their pronounced killing effects to other living cells is still an issue. Active targeting to certain specific receptors in leukaemic cells is the best way to avoid damage to other living cells. Leukaemic cells can be targeted using novel nanoparticles (NPs) coated with a specific ligand, such as octreotide (OCD), to target somatostatin receptor type 2 (SSTR2), which is expressed in leukaemic cells. METHODS: Amino-PEGylated quantum dots (QDs) were chosen as model NPs. The QDs were first succinylated using succinic anhydride and then coated with OCD. The reactivity and selectivity of the formulated QDs-OCD were studied in cell lines with well-expressed SSTR2, while fluorescence was detected using confocal laser scanning microscopy (CLSM) and flow cytometry (FACS). Conclusively, QD-OCD targeting to blood cells was studied in vivo in mice and detected using inductively coupled plasma mass spectrometry and CLSM in tissues. RESULTS: Highly stable QDs coated with OCD were prepared. FACS and CLSM showed highly definite interactions with overexpressed SSTR2 in the investigated cell lines. Moreover, the in vivo results revealed a higher concentration of QDs-OCD in blood cells. The fluorescence intensity of the QDs-OCD was highly accumulated in blood cells, while the unmodified QDs did not accumulate significantly in blood cells. CONCLUSION: The formulated novel QDs-OCD can target SSTR2 overexpressed in blood cells with great potential for treating blood cancer.

Interaction of functionalized nanoparticles with serum proteins and its impact on colloidal stability and cargo leaching.

The majority of effort in the area of polymeric nanocarriers is aimed at providing controlled drug delivery in vivo. Therefore, it is essential to understand the delicate interplay of polymeric NPs with serum proteins in order to forecast their performance in a biological system. In this study, the interaction of serum proteins with functionalized polymeric colloids as a function of particle charge and hydrophobicity was investigated. Moreover, impact on NP stability and cargo leaching was assessed. The hard protein corona of polymeric NPs with either uncharged methoxy groups (methoxy-NPs), positively charged amine groups (amine-NPs), negatively charged carboxylic acid groups (carboxyl-NPs) or zwitterionic NPs decorated with amine and carboxylic acid groups (zwitterion-NPs) was quantitatively and qualitatively analyzed and correlated with the respective colloidal stability using fluorescence resonance energy transfer. Positively charged amine-NPs displayed an enhanced interaction with serum proteins via electrostatic interactions resulting in a hard corona consisting of diverse protein components. As revealed by FRET and agarose gel electrophoresis, the enhanced adsorption of proteins onto the colloidal surface significantly altered the NP identity and severely impaired the colloidal integrity as the lipophilic cargo was continuously leached out of the hydrophobic NP core. These results highlight the importance of generating a profound knowledge of the bio-nano interface as adherence of biomolecules can severely compromise the performance of a colloidal drug delivery system by changing its identity and integrity.

Targeting the Central Nervous System (CNS): A Review of Rabies Virus-Targeting Strategies.

The transport of drugs across the blood-brain barrier is challenging. The use of peptide sequences derived from viruses with a central nervous system (CNS) tropism is one elegant option. A prominent example is the rabies virus glycopeptide-29 (RVG-29), which is said to enable a targeted brain delivery. Although the entry mechanism of the rabies virus into the CNS is very well characterized, it is unknown whether RVG-29-functionalized drug delivery systems (DDSs) follow this pathway. RVG-29-functionalized DDSs present themselves with modifications of the RVG-29 peptide sequence and different physicochemical properties compared to the rabies virus. To our surprise, the impact of these changes on the functionality is completely neglected. This review explores virus-related CNS-targeting strategies by comparing RVG-29-functionalized DDSs with regard to their peptide modification, physiochemical properties and their behavior in cell culture studies with a special focus on the original pathway of rabies virus entry into the CNS.

Controlled Antibody Release from Degradable Thermoresponsive Hydrogels Cross-Linked by Diels-Alder Chemistry.

Amine-modified four- and eight-armed poloxamines were prepared and subsequently functionalized with maleimide or furyl groups. Aqueous solutions of these polymers exhibited an immediate gelation at a temperature above 37 °C. Concomitantly, Diels-Alder reactions gradually cross-linked and cured the gels. Different ratios between four- and eight-armed macromonomers were used to tune hydrogel stability and mechanical properties. In this way, hydrogel stability could be precisely controlled in the range of 14 to 329 days. Controlled release of the model antibody bevacizumab was achieved over a period of 7, 21, and 115 days. Release profiles were triphasic with a low burst; approximately 87% of the released antibody was intact and displayed functional binding. The hydrogels presented in this study are degradable, nontoxic, rapidly gelling, stable, and provide controlled antibody release. They can be tailored to match the demands of various applications and present an attractive platform for antibody delivery.

Ligand-functionalized nanoparticles target endothelial cells in retinal capillaries after systemic application.

To date, diseases affecting vascular structures in the posterior eye are mostly treated by laser photocoagulation and multiple intraocular injections, procedures that destroy healthy tissue and can cause vision-threatening complications. To overcome these drawbacks, we investigate the feasibility of receptor-mediated nanoparticle targeting to capillary endothelial cells in the retina after i.v. application. Cell-binding studies using microvascular endothelial cells showed receptor-specific binding and cellular uptake of cyclo(RGDfC)-modified quantum dots via the αvß3 integrin receptor. Conversely, Mueller cells and astrocytes, representing off-target cells located in the retina, revealed only negligible interaction with nanoparticles. In vivo experiments, using nude mice as the model organism, demonstrated a strong binding of the ligand-modified quantum dots in the choriocapillaris and intraretinal capillaries upon i.v. injection and 1-h circulation time. Nontargeted nanoparticles, in contrast, did not accumulate to a significant amount in the target tissue. The presented strategy of targeting integrin receptors in the retina could be of utmost value for future intervention in pathologies of the posterior eye, which are to date only accessible with difficulty.

Branched Polymer-Drug Conjugates for Multivalent Blockade of Angiotensin II Receptors.

The use of angiotensin receptor blockers (ARBs) for treatment of ocular diseases associated with neovascularizations, such as proliferative diabetic retinopathy, shows tremendous promise but is presently limited due to short intravitreal half-life. Conjugation of ARB molecules to branched polymers could vastly augment their therapeutic efficacy. EXP3174, a potent non-peptide ARB, was conjugated to branched poly(ethylene glycol) (PEG) and poly(amido amine) (PAMAM) dendrimers: 7.8 ligand molecules were tethered to each 40 kDa PEG molecule whereas 16.7 ligand molecules were linked to each PAMAM generation 5 dendrimer. The multivalent PEG and PAMAM conjugates blocked AT1R signaling with an IC50 of 224 and 36.3 nM, respectively. The 6-fold higher affinity of the multivalent ligand-conjugated PAMAM dendrimers was due to their unique microarchitecture and ability to suppress polymer-drug interactions. Remarkably, both polymer-drug conjugates exhibited no cytotoxicity, in stark contrast to plain PAMAM dendrimers. With sufficiently long vitreous half-lives, both synthesized polymer-ARB conjugates have the potential to pave a new path for the therapy of ocular diseases accompanied by retinal neovascularizations.

Diels-Alder Hydrogels for Controlled Antibody Release: Correlation between Mesh Size and Release Rate.

Eight-armed PEG, molecular mass 10 kDa, was functionalized with furyl and maleimide groups, respectively; the obtained macromonomers were cross-linked via Diels-Alder chemistry. The mesh size (ξ) of the prepared hydrogels was determined by swelling studies, rheology, and low field NMR spectroscopy. The in vitro release of fluorescein isothiocyanate labeled dextrans (FDs) and bevacizumab was investigated. The average mesh size (ξavg) increased from 5.8 ± 0.1 nm to 56 ± 13 nm during degradation, as determined by swelling studies. The result of the rheological measurements (8.0 nm) matched the initial value of ξavg. Low field NMR spectroscopy enabled the determination of the mesh size distribution; the most abundant mesh size was found to be 9.2 nm. In combination with the hydrodynamic radius of the molecule (Rh), the time-dependent increase of ξavg was used to predict the release profiles of incorporated FDs applying an obstruction-scaling model. The predicted release profiles matched the experimentally determined release profiles when Rh < ξavg. However, significant deviations from the theoretical predictions were observed when Rh ≥ ξavg, most likely due to the statistical distribution of ξ in real polymer networks. The release profile of bevacizumab differed from those of equivalently sized FDs. The delayed release of bevacizumab was most likely a result of the globular structure and rigidity of the protein. The observed correlation between ξ and the release rate could facilitate the design of controlled release systems for antibodies.

On the uncertainty of the correlation between nanoparticle avidity and biodistribution.

The specific delivery of a drug to its site of action also known as targeted drug delivery is a topic in the field of pharmaceutics studied for decades. One approach extensively investigated in this context is the use ligand functionalized nanoparticles. These particles are modified to carry receptor specific ligands, enabling them to accumulate at a desired target site. However, while this concept initially appears straightforward to implement, in-depth research has revealed several challenges hindering target site specific particle accumulation - some of which remain unresolved to this day. One of these challenges consists in the still incomplete understanding of how nanoparticles interact with biological systems. This knowledge gap significantly compromises the predictability of particle distribution in biological systems, which is critical for therapeutic efficacy. One of the most crucial steps in delivery is the attachment of nanoparticles to cells at the target site. This attachment occurs via the formation of multiple ligand receptor bonds. A process also referred to as multivalent interaction. While multivalency has been described extensively for individual molecules and macromolecules respectively, little is known on the multivalent binding of nanoparticles to cells. Here, we will specifically introduce the concept of avidity as a measure for favorable particle membrane interactions. Also, an overview about nanoparticle and membrane properties affecting avidity will be given. Thereafter, we provide a thorough review on literature investigating the correlation between nanoparticle avidity and success in targeted particle delivery. In particular, we want to analyze the currently uncertain data on the existence and nature of the correlation between particle avidity and biodistribution.

Conditional Cell-Penetrating Peptide Exposure as Selective Nanoparticle Uptake Signal.

A major bottleneck diminishing the therapeutic efficacy of various drugs is that only small proportions of the administered dose reach the site of action. One promising approach to increase the drug amount in the target tissue is the delivery via nanoparticles (NPs) modified with ligands of cell surface receptors for the selective identification of target cells. However, since receptor binding can unintentionally trigger intracellular signaling cascades, our objective was to develop a receptor-independent way of NP uptake. Cell-penetrating peptides (CPPs) are an attractive tool since they allow efficient cell membrane crossing. So far, their applicability is severely limited as their uptake-promoting ability is nonspecific. Therefore, we aimed to achieve a conditional CPP-mediated NP internalization exclusively into target cells. We synthesized different CPP candidates and investigated their influence on nanoparticle stability, ζ-potential, and uptake characteristics in a core-shell nanoparticle system consisting of poly(lactid-co-glycolid) (PLGA) and poly(lactic acid)-poly(ethylene glycol) (PLA10kPEG2k) block copolymers with CPPs attached to the PEG part. We identified TAT47-57 (TAT) as the most promising candidate and subsequently combined the TAT-modified PLA10kPEG2k polymer with longer PLA10kPEG5k polymer chains, modified with the potent angiotensin-converting enzyme 2 (ACE2) inhibitor MLN-4760. While MLN-4760 enables selective target cell identification, the additional PEG length hides the CPP during a first unspecific cell contact. Only after the previous selective binding of MLN-4760 to ACE2, the established spatial proximity exposes the CPP, triggering cell uptake. We found an 18-fold uptake improvement in ACE2-positive cells compared to unmodified particles. In summary, our work paves the way for a conditional and thus highly selective receptor-independent nanoparticle uptake, which is beneficial in terms of avoiding side effects.

Prolonged delivery of HIV-1 vaccine nanoparticles from hydrogels.

Immunization is a straightforward concept but remains for some pathogens like HIV-1 a challenge. Thus, new approaches towards increasing the efficacy of vaccines are required to turn the tide. There is increasing evidence that antigen exposure over several days to weeks induces a much stronger and more sustained immune response compared to traditional bolus injection, which usually leads to antigen elimination from the body within a couple of days. Therefore, we developed a poly(ethylene) glycol (PEG) hydrogel platform to investigate the principal feasibility of a sustained release of antigens to mimic natural infection kinetics. Eight-and four-armed PEG macromonomers of different MWs (10, 20, and 40 kDa) were end-group functionalized to allow for hydrogel formation via covalent cross-linking. An HIV-1 envelope (Env) antigen in its trimeric (Envtri) or monomeric (Envmono) form was applied. The soluble Env antigen was compared to a formulation of Env attached to silica nanoparticles (Env-SiNPs). The latter are known to have a higher immunogenicity compared to their soluble counterparts. Hydrogels were tunable regarding the rheological behavior allowing for different degradation times and release timeframes of Env-SiNPs over two to up to 50 days. Affinity measurements of the VCR01 antibody which specifically recognizes the CD4 binding site of Env, revealed that neither the integrity nor the functionality of Envmono-SiNPs (Kd = 2.1 ± 0.9 nM) and Envtri-SiNPs (Kd = 1.5 ± 1.3 nM), respectively, were impaired after release from the hydrogel (Kd before release: 2.1 ± 0.1 and 7.8 ± 5.3 nM, respectively). Finally, soluble Env and Env-SiNPs which are two physico-chemically distinct compounds, were co-delivered and shown to be sequentially released from one hydrogel which could be beneficial in terms of heterologous immunization or single dose vaccination. In summary, this study presents a tunable, versatile applicable, and effective delivery platform that could improve vaccination effectiveness also for other infectious diseases than HIV-1.

Drug loading of polymeric micelles.

PURPOSE: To gain mechanistic insights into drug loading and lyophilization of polymeric micelles. METHODS: PEGylated poly-4-(vinylpyridine) micelles were loaded with dexamethasone. Three different methods were applied and compared: O/W emulsion, direct dialysis, cosolvent evaporation. Micellar dispersions with the highest drug load were lyophilized with varying lyoprotectors: sucrose, trehalose, maltose, a polyvinylpyrrolidine derivative, and ß-cyclodextrin derivatives. For comparison, other PEGylated block copolymer micelles (PEGylated polylactic acid, polylactic acid-co-glycolic acid, polycaprolactone) were freeze-dried. RESULTS: Drug loading via direct dialysis from acetone was a less effective loading method which led to dexamethasone loads <2% w/w. O/W emulsion technique from dichlormethane increased drug load up to ~13% w/w; optimized cosolvent evaporation increased load up to ~19% w/w. An important step for cosolvent evaporation was solubility screen of the drug prior to preparation. Loading was maintained upon lyophilization with ß-cyclodextrins which proved to be versatile stabilizers for other block copolymer micelles. CONCLUSION: Careful solvent selection prior to cosolvent evaporation was a beneficial approach to load hydrophobic drugs into polymeric micelles. Moreover, ß-cyclodextrins could be used as versatile lyoprotectors for these micelles.

Long time monitoring of the respiratory activity of isolated mitochondria.

We have investigated the ability of optical oxygen sensors incorporated in a microplate to determine the respiratory activity of cell fractions. Different cell fractions were monitored, in particular to evaluate the long term functionality of isolated mitochondria. It is possible to continuously sense respiratory activity of isolated mitochondria over time. We found that they are functional for three hours but stop respiring at a critical limit of 20% air saturation in the system. Furthermore, inhibition and enhancement of respiratory activity were detected. In conclusion, oxygen sensors are a powerful tool to evaluate the functionality of isolated mitochondria.

G protein-coupled receptors function as logic gates for nanoparticle binding and cell uptake.

More selective interactions of nanoparticles with cells would substantially increase their potential for diagnostic and therapeutic applications. Thus, it would not only be highly desirable that nanoparticles can be addressed to any cell with high target specificity and affinity, but that we could unequivocally define whether they rest immobilized on the cell surface as a diagnostic tag, or if they are internalized to serve as a delivery vehicle for drugs. To date no class of targets is known that would allow direction of nanoparticle interactions with cells alternatively into one of these mutually exclusive events. Using MCF-7 breast cancer cells expressing the human Y(1)-receptor, we demonstrate that G protein-coupled receptors provide us with this option. We show that quantum dots carrying a surface-immobilized antagonist remain with nanomolar affinity on the cell surface, and particles carrying an agonist are internalized upon receptor binding. The receptor functions like a logic "and-gate" that grants cell access only to those particles that carry a receptor ligand "and" where the ligand is an agonist. We found that agonist- and antagonist-modified nanoparticles bind to several receptor molecules at a time. This multiligand binding leads to five orders of magnitude increased-receptor affinities, compared with free ligand, in displacement studies. More than 800 G protein-coupled receptors in humans provide us with the paramount advantage that targeting of a plethora of cells is possible, and that switching from cell recognition to cell uptake is simply a matter of nanoparticle surface modification with the appropriate choice of ligand type.

Insulin is essential for in vitro chondrogenesis of mesenchymal progenitor cells and influences chondrogenesis in a dose-dependent manner.

PURPOSE: Insulin is a commonly used additive in chondrogenic media for differentiating mesenchymal stem cells (MSCs). The indispensability of other bioactive factors like TGF-ß or dexamethasone in these medium formulations has been shown, but the role of insulin is unclear. The purpose of this study was to investigate whether insulin is essential for MSC chondrogenesis and if there is a dose-dependent effect of insulin on MSC chondrogenesis. METHODS: We cultivated human MSCs in pellet culture in serum-free chondrogenic medium with insulin concentrations between 0 and 50 µg/ml and assessed the grade of chondrogenic differentiation by histological evaluation and determination of glycosaminoglycan (GAG), total collagen and DNA content. We further tested whether insulin can be delivered in an amount sufficient for MSC chondrogenesis via a drug delivery system in insulin-free medium. RESULTS: Chondrogenesis was not induced by standard chondrogenic medium without insulin and the expression of cartilage differentiation markers was dose-dependent at insulin concentrations between 0 and 10 µg/ml. An insulin concentration of 50 µg/ml had no additional effect compared with 10 µg/ml. Insulin was delivered by a release system into the cell culture under insulin-free conditions in an amount sufficient to induce chondrogenesis. CONCLUSIONS: Insulin is essential for MSC chondrogenesis in this system and chondrogenic differentiation is influenced by insulin in a dose-dependent manner. Insulin can be provided in a sufficient amount by a drug delivery system. Therefore, insulin is a suitable and inexpensive indicator substance for testing drug release systems in vitro.