Maleimide-Decorated PEGylated Mucoadhesive Liposomes for Ocular Drug Delivery.

Liposomes are promising spherical vesicles for topical drug delivery to the eye. Several types of vesicles were formulated in this study, including conventional, PEGylated, and maleimide-decorated PEGylated liposomes. The physicochemical characteristics of these liposomes, including their size, zeta potential, ciprofloxacin encapsulation efficiency, loading capacity, and release, were evaluated. The structure of these liposomes was examined using dynamic light scattering, transmission electron microscopy, and small angle neutron scattering. The ex vivo corneal and conjunctival retention of these liposomes were examined using the fluorescence flow-through method. Maleimide-decorated liposomes exhibited the best retention performance on bovine conjunctiva compared to other types of liposomes studied. Poor retention of all liposomal formulations was observed on bovine cornea.

Fluorine-Containing Block and Gradient Copoly(2-oxazoline)s Based on 2-(3,3,3-Trifluoropropyl)-2-oxazoline: A Quest for the Optimal Self-Assembled Structure for 19F Imaging.

The use of fluorinated contrast agents in magnetic resonance imaging (MRI) facilitates improved image quality due to the negligible amount of endogenous fluorine atoms in the body. In this work, we present a comprehensive study of the influence of the amphiphilic polymer structure and composition on its applicability as contrast agents in 19F MRI. Three series of novel fluorine-containing poly(2-oxazoline) copolymers and terpolymers, hydrophilic-fluorophilic, hydrophilic-lipophilic-fluorophilic, and hydrophilic-thermoresponsive-fluorophilic, with block and gradient distributions of the fluorinated units, were synthesized. It was discovered that the CF3 in the 2-(3,3,3-trifluoropropyl)-2-oxazoline (CF3EtOx) group activated the cationic chain end, leading to faster copolymerization kinetics, whereby spontaneous monomer gradients were formed with accelerated incorporation of 2-methyl-2-oxazoline or 2-n-propyl-2-oxazoline with a gradual change to the less-nucleophilic CF3EtOx monomer. The obtained amphiphilic copolymers and terpolymers form spherical or wormlike micelles in water, which was confirmed using transmission electron microscopy (TEM), while small-angle X-ray scattering (SAXS) revealed the core-shell or core-double-shell morphologies of these nanoparticles. The core and shell sizes obey the scaling laws for starlike micelles predicted by the scaling theory. Biocompatibility studies confirm that all copolymers obtained are noncytotoxic and, at the same time, exhibit high sensitivity during in vitro 19F MRI studies. The gradient copolymers provide the best 19F MRI signal-to-noise ratio in comparison with the analogue block copolymer structures, making them most promising as 19F MRI contrast agents.

Polymer Cancerostatics Targeted by Recombinant Antibody Fragments to GD2-Positive Tumor Cells.

A water-soluble polymer cancerostatic actively targeted against cancer cells expressing a disialoganglioside antigen GD2 was designed, synthesized and characterized. A polymer conjugate of an antitumor drug doxorubicin with a N-(2-hydroxypropyl)methacrylamide-based copolymer was specifically targeted against GD2 antigen-positive tumor cells using a recombinant single chain fragment (scFv) of an anti-GD2 monoclonal antibody. The targeting protein ligand was attached to the polymer-drug conjugate either via a covalent bond between the amino groups of the protein using a traditional nonspecific aminolytic reaction with a reactive polymer precursor or via a noncovalent but highly specific interaction between bungarotoxin covalently linked to the polymer and the recombinant scFv modified with a C-terminal bungarotoxin-binding peptide. The GD2 antigen binding activity and GD2-specific cytotoxicity of the targeted noncovalent polymer-scFv complex proved to be superior to the covalent polymer-scFv conjugate.

Binding of HSA to Macromolecular pHPMA Based Nanoparticles for Drug Delivery: An Investigation Using Fluorescence Methods.

Amphiphilic poly( N-(2-hydroxypropyl)methacrylamide) copolymers ( pHPMA) bearing cholesterol side groups in phosphate buffer saline self-assemble into nanoparticles (NPs) which can be used as tumor-targeted drug carriers. It was previously shown by us that human serum albumin (HSA) interacts weakly with the NPs. However, the mechanism of this binding could not be resolved due to overlapping of signals from the complex system. Here, we use fluorescence labeling to distinguish the components and to characterize the binding: On the one hand, a fluorescent dye was attached to pHPMA, so that the diffusion behavior of the NPs could be studied in the presence of HSA using fluorescence lifetime correlation spectroscopy. On the other hand, quenching of the intrinsic fluorescence of HSA revealed the origin of the binding, which is mainly the complexation between HSA and cholesterol side groups. Furthermore, a binding constant was obtained.

Macromolecular pHPMA-Based Nanoparticles with Cholesterol for Solid Tumor Targeting: Behavior in HSA Protein Environment.

Nanoparticles (NPs) that form by self-assembly of amphiphilic poly(N-(2-hydroxypropyl)-methacrylamide) (pHPMA) copolymers bearing cholesterol side groups are potential drug carriers for solid tumor treatment. Here, we investigate their behavior in solutions of human serum albumin (HSA) in phosphate buffered saline. Mixed solutions of NPs, from polymer conjugates with or without the anticancer drug doxorubicin (Dox) bound to them, and HSA at concentrations up to the physiological value are characterized by synchrotron small-angle X-ray scattering and isothermal titration calorimetry. When Dox is absent, a small amount of HSA molecules bind to the cholesterol groups that form the core of the NPs by diffusing through the loose pHPMA shell or get caught in meshes formed by the pHPMA chains. These interactions are strongly hindered by the presence of Dox, which is distributed in the pHPMA shell, meaning that the delivery of Dox by the NPs in the human body is not affected by the presence of HSA.

Formation of core/corona nanoparticles with interpolyelectrolyte complex cores in aqueous solution: insight into chain dynamics in the complex from fluorescence quenching.

Formation of interpolyelectrolyte complexes (IPECs) of poly(methacrylic acid) (PMAA) bearing a fluorescent label (umbelliferone) at the chain end and poly[3,5-bis(trimethyl ammoniummethyl)-4-hydroxystyrene iodide]-block-poly(ethylene oxide) (QNPHOS-PEO) acting as a fluorescence quencher, was followed using a combination of scattering, calorimetry, microscopy and fluorescence spectroscopy techniques. While scattering and microscopy measurements indicated formation of spherical core/corona nanoparticles with the core of the QNPHOS/PMAA complex and the PEO corona, fluorescence measurements showed that both static and dynamic quenching efficiency were increased in the nanoparticle stability region. As the dynamic quenching rate constant remained unchanged, the quenching enhancement was caused by the increase in the local concentration of QNPHOS segments in the microenvironment of the label. This finding implies that the local dynamics of PMAA end chains affecting the interaction of the label with QNPHOS segments was independent of both PMAA and QNPHOS chain conformations.

Polyelectrolyte pH-Responsive Protein-Containing Nanoparticles: The Physicochemical Supramolecular Approach.

We report on the physicochemical properties and self-assembly behavior of novel efficient pH-sensitive nanocontainers based on the Food and Drug Administration-approved anionic polymer Eudragit L100-55 (poly(methacrylic acid-co-ethyl acrylate) 1:1) and nonionic surfactant Brij98. The features of the interaction between Eudragit L100-55 and Brij98 at different pH values and their optimal ratio for nanoparticle formation were studied using isothermal titration calorimetry. The influence of the polymer-to-surfactant ratio on the size and structure of particles was studied at different pH values using dynamic light scattering and small-angle X-ray scattering methods. It was shown that stable nanoparticles are formed at acidic pH at polymer-to-surfactant molar ratios from 1:43 to 1:139. Trypsin was successfully encapsulated into Eudragit-Brij98 nanoparticles as a model bioactive component. The loading efficiency was determined by labeling trypsin with radioactive iodine-125. Eudragit-Brij98 nanoparticles effectively protected trypsin against pepsin digestion. The results showed that trypsin encapsulated into novel pH-sensitive nanocontainers retained more than 50% of its activity after treatment with pepsin compared with nonencapsulated trypsin. The described concept will contribute both to understanding the principles of and designing next-generation nanocontainers.

pH Responsiveness of hydrogels formed by telechelic polyampholytes.

We investigate the influence of pH on the rheological and structural properties of hydrogels formed by hydrophobic association of the sticky ends of the triblock terpolymer poly(methyl methacrylate)-b-poly(2-(diethylamino)ethyl methacrylate-co-methacrylic acid)-b-poly(methyl methacrylate) (PMMA-b-P(DEA-co-MAA)-b-PMMA). The middle block is a weak polyampholyte having a pH dependent charge density and sign, which enables tuning of the rheological and structural properties by pH variation. Small-angle neutron scattering (SANS) studies of solutions in D2O at 0.05 wt% and pH 3.0 reveal clusters of interconnected spherical micelles having PMMA cores, stabilized by repulsive ionic interactions in the middle polyampholyte block. With increasing pH, the degree of ionization of the DEA units decreases, whereas the one of the MAA units increases, resulting in a complete loss of the correlation between these micelles. At a concentration of 3 wt% at low pH values, the system forms a gel with charged fuzzy spheres from PMMA interacting via a screened Coulomb potential. With increasing pH, the gel disintegrates due to the decrease in the effective charge on the micelles. At both concentrations, the hydrophobic aggregation of micelles is observed near the isoelectric point. At pH 3.0-7.4, the autocorrelation functions measured by rotational dynamic light scattering at 3 wt% exhibit a decay steeper than single exponential, which confirms that the gels are frozen, presumably due to the glassy PMMA cores and hydrophobic interpolyelectrolyte complexes. At pH 11, the diffusion of single micelles is observed in addition to the frozen dynamics.

System with embedded drug release and nanoparticle degradation sensor showing efficient rifampicin delivery into macrophages.

We have developed a biodegradable, biocompatible system for the delivery of the antituberculotic antibiotic rifampicin with a built-in drug release and nanoparticle degradation fluorescence sensor. Polymer nanoparticles based on poly(ethylene oxide) monomethyl ether-block-poly(ε-caprolactone) were noncovalently loaded with rifampicin, a combination that, to best of our knowledge, was not previously described in the literature, which showed significant benefits. The nanoparticles contain a Förster resonance energy transfer (FRET) system that allows real-time assessment of drug release not only in vitro, but also in living macrophages where the mycobacteria typically reside as hard-to-kill intracellular parasites. The fluorophore also enables in situ monitoring of the enzymatic nanoparticle degradation in the macrophages. We show that the nanoparticles are efficiently taken up by macrophages, where they are very quickly associated with the lysosomal compartment. After drug release, the nanoparticles in the cmacrophages are enzymatically degraded, with half-life 88±11 min.

Synthesis and supramolecular properties of regioisomers of mononaphthylallyl derivatives of γ-cyclodextrin.

Monosubstituted derivatives of γ-cyclodextrin (γ-CD) are suitable building blocks for supramolecular polymers, and can also serve as precursors for the synthesis of other regioselectively monosubstituted γ-CD derivatives. We prepared a set of monosubstituted 2I-O-, 3I-O-, and 6I-O-(3-(naphthalen-2-yl)prop-2-en-1-yl) derivatives of γ-CD using two different methods. A key step of the first synthetic procedure is a cross-metathesis between previously described regioisomers of mono-O-allyl derivatives of γ-CD and 2-vinylnaphthalene which gives yields of about 16-25% (2-5% starting from γ-CD). To increase the overall yields, we have developed another method, based on a direct alkylation of γ-CD with 3-(naphthalen-2-yl)allyl chloride as the alkylating reagent. Highly regioselective reaction conditions, which differ for each regioisomer in a used base, gave the monosubstituted isomers in yields between 12-19%. Supramolecular properties of these derivatives were studied by DLS, ITC, NMR, and Cryo-TEM.

Internal Nanoparticle Structure of Temperature-Responsive Self-Assembled PNIPAM-b-PEG-b-PNIPAM Triblock Copolymers in Aqueous Solutions: NMR, SANS, and Light Scattering Studies.

In this study, we report detailed information on the internal structure of PNIPAM-b-PEG-b-PNIPAM nanoparticles formed from self-assembly in aqueous solutions upon increase in temperature. NMR spectroscopy, light scattering, and small-angle neutron scattering (SANS) were used to monitor different stages of nanoparticle formation as a function of temperature, providing insight into the fundamental processes involved. The presence of PEG in a copolymer structure significantly affects the formation of nanoparticles, making their transition to occur over a broader temperature range. The crucial parameter that controls the transition is the ratio of PEG/PNIPAM. For pure PNIPAM, the transition is sharp; the higher the PEG/PNIPAM ratio results in a broader transition. This behavior is explained by different mechanisms of PNIPAM block incorporation during nanoparticle formation at different PEG/PNIPAM ratios. Contrast variation experiments using SANS show that the structure of nanoparticles above cloud point temperatures for PNIPAM-b-PEG-b-PNIPAM copolymers is drastically different from the structure of PNIPAM mesoglobules. In contrast with pure PNIPAM mesoglobules, where solidlike particles and chain network with a mesh size of 1-3 nm are present, nanoparticles formed from PNIPAM-b-PEG-b-PNIPAM copolymers have nonuniform structure with "frozen" areas interconnected by single chains in Gaussian conformation. SANS data with deuterated "invisible" PEG blocks imply that PEG is uniformly distributed inside of a nanoparticle. It is kinetically flexible PEG blocks which affect the nanoparticle formation by prevention of PNIPAM microphase separation.

Biodegradable Micellar HPMA-Based Polymer-Drug Conjugates with Betulinic Acid for Passive Tumor Targeting.

Here, we present the synthesis, physicochemical, and preliminary biological characterization of micellar polymer-betulinic acid (BA) conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carriers, enabling the controlled release of cytotoxic BA derivatives in solid tumors or tumor cells. Various HPMA copolymer conjugates differing in the structure of the spacer between the drug and the carrier were synthesized, all designed for pH-triggered drug release in tumor tissue or tumor cells. The high molecular weight of the micellar conjugates should improve the uptake of the drug in solid tumors due to the Enhanced permeability and retention (EPR) effect. Nevertheless, only the conjugate containing BA with methylated carboxyl groups enabled pH-dependent controlled release in vitro. Moreover, drug release led to the disassembly of the micellar structure, which facilitated elimination of the water-soluble HPMA copolymer carrier from the body by renal filtration. The methylated BA derivative and its polymer conjugate exhibited high cytostatic activity against DLD-1, HT-29, and HeLa carcinoma cell lines and enhanced tumor accumulation in HT-29 xenograft in mice.

Thermodynamics of the multi-stage self-assembly of pH-sensitive gradient copolymers in aqueous solutions.

The self-assembly thermodynamics of pH-sensitive di-block and tri-block gradient copolymers of acrylic acid and styrene was studied for the first time using isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) performed at varying pH. We were able to monitor each step of micellization as a function of decreasing pH. The growth of micelles is a multi-stage process that is pH dependent with several exothermic and endothermic components. The first step of protonation of the acrylic acid monomer units was accompanied mainly by conformational changes and the beginning of self-assembly. In the second stage of self-assembly, the micelles become larger and the number of micelles becomes smaller. While solution acidity increases, the isothermal calorimetry data show a broad deep minimum corresponding to an exothermic process attributed to an increase in the size of hydrophobic domains and an increase in the structure's hydrophobicity. The minor change in heat capacity (ΔCp) confirms the structural changes during this exothermic process. The exothermic process terminates deionization of acrylic acid. The pH-dependence of the ζ-potential of the block gradient copolymer micelles exhibits a plateau in the regime corresponding to the pH-controlled variation of the micellar dimensions. The onset of micelle formation and the solubility of the gradient copolymers were found to be dependent on the length of the gradient block.

Thermoresponsive Polymer Micelles as Potential Nanosized Cancerostatics.

An effective chemotherapy for neoplastic diseases requires the use of drugs that can reach the site of action at a therapeutically efficacious concentration and maintain it at a constant level over a sufficient period of time with minimal side effects. Currently, conjugates of high-molecular-weight hydrophilic polymers or biocompatible nanoparticles with stimuli-releasable anticancer drugs are considered to be some of the most promising systems capable of fulfilling these criteria. In this work, conjugates of thermoresponsive diblock copolymers with the covalently bound cancerostatic drug pirarubicin (PIR) were synthesized as a reversible micelle-forming drug delivery system combining the benefits of the above-mentioned carriers. The diblock copolymer carriers were composed of hydrophilic poly[N-(2-hydroxypropyl)methacrylamide]-based block containing a small amount (∼ 5 mol %) of comonomer units with reactive hydrazide groups and a thermoresponsive poly[2-(2-methoxyethoxy)ethyl methacrylate] block. PIR was attached to the hydrophilic block of the copolymer through the pH-sensitive hydrazone bond designed to be stable in the bloodstream at pH 7.4 but to be degraded in an intratumoral/intracellular environment at pH 5-6. The temperature-induced conformation change of the thermoresponsive block (coil-globule transition), followed by self-assembly of the copolymer into a micellar structure, was controlled by the thermoresponsive block length and PIR content. The cytotoxicity and intracellular transport of the conjugates as well as the release of PIR from the conjugates inside the cells, followed by its accumulation in the cell nuclei, were evaluated in vitro using human colon adenocarcinoma (DLD-1) cell lines. It was demonstrated that the studied conjugates have a great potential to become efficacious in vivo pharmaceuticals.

Formation of DNA-copolymer fibrils through an amyloid-like nucleation polymerization mechanism.

Conjugation of a hydrophobic poly(2-oxazoline) bearing tertiary amide groups along its backbone with a short single stranded nucleotide sequence results in an amphiphilic comb/graft copolymer, which organizes in fibrils upon direct dissolution in water. Supported by circular dichroism, atomic force microscopy, transmission electron microscopy, and scattering data, fibrils are formed through inter- and intramolecular hydrogen bonding between hydrogen accepting amide groups along the polymer backbone and hydrogen donating nucleic acid grafts leading to the formation of hollow tubes.

Properties of cationic monosubstituted tetraalkylammonium cyclodextrin derivatives - their stability, complexation ability in solution or when deposited on solid anionic surface.

The thermal stability of the monosubstituted cationic cyclodextrin (CD) derivatives PEMEDA-ß-CD and PEMPDA-ß-CD, which differ in their substituent linker length (ethylene and propylene, respectively), was studied via (1)H NMR experiments. PEMPDA-ß-CD exhibited higher resistance towards the Hofmann degradation and was chosen as a more suitable host molecule for further studies. Inclusion properties of PEMPDA-ß-CD in solution with a series of simple aromatic guests (salicylic acid, p-methoxyphenol and p-nitroaniline) were determined by isothermal titration calorimetry (ITC) and compared to the native ß-CD. Permanently charged cationic CD derivatives were successfully deposited on the anionic solid surface of polymeric Nafion(®) 117 membrane via electrostatic interactions. Deposition kinetics and coverage of the surface were determined by ELSD. Finally, the ability of the CD derivatives bound to the solid surface to encapsulate aromatic compounds from aqueous solution was measured by UV-vis spectroscopy. The obtained results are promising for future industrial applications of the monosubstituted ß-CD derivatives, because the preparation of cationic CD derivatives is applicable in large scale, without the need of chromatographic purification. Their ionic deposition on a solid surface is simple, yet robust and a straightforward process as well.

Self-assembly thermodynamics of pH-responsive amino-acid-based polymers with a nonionic surfactant.

The behavior of pH-responsive polymers poly(N-methacryloyl-l-valine) (P1), poly(N-methacryloyl-l-phenylalanine) (P2), and poly(N-methacryloylglycyne-l-leucine) (P3) has been studied in the presence of the nonionic surfactant Brij98. The pure polymers phase-separate in an acidic medium with critical pHtr values of 3.7, 5.5, and 3.4, respectively. The addition of the surfactant prevents phase separation and promotes reorganization of polymer molecules. The nature of the interaction between polymer and surfactant depends on the amino acid structure in the side chain of the polymer. This effect was investigated by dynamic light scattering, isothermal titration calorimetry, electrophoretic measurements, small-angle neutron scattering, and infrared spectroscopy. Thermodynamic analysis revealed an endothermic association reaction in P1/Brij98 mixture, whereas a strong exothermic effect was observed for P2/Brij98 and P3/Brij98. Application of regular solution theory for the analysis of experimental enthalpograms indicated dominant hydrophobic interactions between P1 and Brij98 and specific interactions for the P2/Brij98 system. Electrophoretic and dynamic light scattering measurements support the applicability of the theory to these cases. The specific interactions can be ascribed to hydrogen bonds formed between the carboxylic groups of the polymer and the oligo(ethylene oxide) head groups of the surfactant. Thus, differences in polymer-surfactant interactions between P1 and P2 polymers result in different structures of polymer-surfactant complexes. Specifically, small-angle neutron scattering revealed pearl-necklace complexes and "core-shell" structures for P1/Brij98 and P2/Brij98 systems, respectively. These results may help in the design of new pH-responsive site-specific micellar drug delivery systems or pH-responsive membrane-disrupting agents.

Coiled coil peptides and polymer-peptide conjugates: synthesis, self-assembly, characterization and potential in drug delivery systems.

Coiled coils are a common structural motif in many natural proteins that can also be utilized in the design and preparation of drug delivery systems for the noncovalent connection of two macromolecules. In this work, two different pairs of peptides forming coiled coil hetero-oligomers were designed, synthesized, and characterized. While the peptide sequences (VAALEKE)4 and (VAALKEK)4 predominantly form coiled coil heterodimers with randomly orientated peptide chains, (IAALESE)2-IAALESKIAALESE and IAALKSKIAALKSE-(IAALKSK)2 tend to form higher hetero-oligomers with an antiparallel orientation of their peptide chains. The associative behavior of these peptides was studied in aqueous solutions using circular dichroism spectroscopy, size-exclusion chromatography, isothermal titration calorimetry and sedimentation analyses. The orientation of the peptide chains in the coiled coil heterodimers was assessed using fluorescence spectroscopy with fluorescence resonance energy transfer labels attached to the ends of the peptides. The formation of the heterodimer can be used as a general method for the selective noncovalent conjugation of a specific targeting moiety with various drug carrier systems; this process involves simple self-assembly in a physiological solution before drug administration. The preparation of targeted macromolecular therapeutics consisting of a synthetic polymer drug carrier and a recombinant protein targeting ligand is discussed.

On the mucoadhesive properties of synthetic and natural polyampholytes.

HYPOTHESIS: The mucoadhesive characteristics of amphoteric polymers (also known as polyampholytes) can vary and are influenced by factors such as the solution's pH and its relative position against their isoelectric point (pHIEP). Whilst the literature contains numerous reports on mucoadhesive properties of either cationic or anionic polymers, very little is known about these characteristics for polyampholytes EXPERIMENTS: Here, two amphoteric polymers were synthesized by reaction of linear polyethylene imine (l-PEI) with succinic or phthalic anhydride and their mucoadhesive properties were compared to bovine serum albumin (BSA), selected as a natural polyampholyte. Interactions between these polymers and porcine gastric mucin were studied using turbidimetric titration and isothermal titration calorimetry across a wide range of pHs. Model tablets were designed, coated with these polymers and tested to evaluate their adhesion to porcine gastric mucosa at different pHs. Moreover, a retention study using fluorescein isothiocyanate (FITC)-labelled polyampholytes deposited onto mucosal surfaces was also conducted FINDINGS: All these studies indicated the importance of solution pH and its relative position against pHIEP in the mucoadhesive properties of polyampholytes. Both synthetic and natural polyampholytes exhibited strong interactions with mucin and good mucoadhesive properties at pH < pHIEP.

Hydrolytically degradable polymer micelles for drug delivery: a SAXS/SANS kinetic study.

We report kinetic studies of therapeutically highly potent polymer-drug conjugates consisting of amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers bearing the anticancer drug doxorubicin (Dox). Highly hydrophobic cholesterol moieties as well as the drug were attached to the polymer backbone by a pH-sensitive hydrazone bond. Moreover, the structure of the spacer between the polymer carrier and the cholesterol moiety differed in order to influence the release rate of the hydrophobic moiety, and thus the disintegration of the high-molecular-weight micellar nanoparticle structure. We performed time-dependent SAXS/SANS measurements after changing pH from a typical blood value (pH 7.2) to that of tumor cells (pH 5.0) to characterize the drug release and changes in particle size and shape. Nanoparticles composed of the conjugates containing Dox were generally larger than the drug-free ones. For most conjugates, nanoparticle growth or decay was observed in the time range of several hours. It was established that the growth/decay rate and the steady-state size of nanoparticles depend on the spacer structure. From analytical fitting, we conclude that the most probable structure of the nanoparticles was a core-shell or a core with attached Gaussian chains. We concluded that the spacer structure determined the fate of a cholesterol derivative after the pH jump. Fitting results for 5α-cholestan-3-onecholestan-3-one and cholesteryl-4-oxopentanoate (Lev-chol) implied that cholesterol moieties continuously escape from the core of the nanoparticle core and concentrate in the hydrophilic shell. In contrast, cholest-4-en-3-one spacer prevent cholesterol escaping. Dox moiety release was only observed after a change in pH. Such findings justify the model proposed in our previous paper. Lastly, the cholesteryl 4-(2-oxopropyl)benzoate (Opb-Chol) was a different case where after the release of hydrophobic Opb-Chol moieties, the core becomes more compact. The physicochemical mechanisms responsible for the scenarios of the different spacers are discussed.