**5. Apo-B100 is a flexible string wrapped around the surface of LDL**

As already indicated above, the physicochemical state of the core lipids is intimately related to the structure and dynamics of the particle surface, which consists of about 700 phospholipid molecules and one single copy of apo-B100. Apo-B100 is a huge glycoprotein and its polypeptide chain consists of 4536 amino acid residues with an estimated molecular mass of about 550 kDa for the glycosilated form [52,53]. Apo-B100 is a single chain protein with a total contour length of about 70 nm [54] and can be viewed as a highly flexible molecular string composed of single domains [20]. Five consecutive domains were identified based on secondary structure elements representing the main conformational motifs of apo-B100. The single domains are connected by flexible interdomain linker regions, which allow relative movements of domains to each other. The feasibility of such motions was shown in a low resolution model of detergent solubilized apo-B100, which was derived from small angle neutron scattering data [55]. In this model, compact rigid domains are visible being connected by flexible interdomain linkages, which possess a substantial degree of freedom in their spatial orientation. A hypothetical spatial arrangement of the apo-B100 molecule on a spherical LDL particle was created after assigning the secondary structure elements, which were deduced from a secondary structure prediction, to the surface of apo-B100 (Figure 4). Likewise, the averaged surface shape of the 3D-model would allow for variations in the thickness of the apo-B100 molecule by about 1 nm. Such variations are most likely required to compensate for changes in the surface area upon lipid exchange and particle shrinking during endogeneous lipoprotein conversion from very low density lipoprotein (VLDL) to LDL.

**Figure 4.** Reconstituted low resolution model of lipid-free apo-B100 derived from small angle neutron scattering data. Apo-B100 shows an elongated arch-like morphology indicating single domains and mobile less defined linker regions. A hypothetical model of a spherical LDL particle after superposition of the structural model of apo-B100 is shown (adapted from ref. [55]). Secondary structure modules are assigned to the surface after a secondary structure prediction was performed. The results nicely correspond to the pentapartite model suggested by [20].

Concerning the topology of apo-B100 on the surface of LDL the most detailed information is obtained from cryo-e.m. images (see also Figure 2). Chatterton et al. were among the first to visualize apo-B100 as string circumventing LDL, and to report on mapped epitopes of apo-B100 distributed over one hemisphere of the LDL particle [56,57]. Recent single particle 3Dreconstructions from immuno cryo e.m. images delineated a more accurate picture of apo-B100 revealing a looped topology of the protein backbone with distinct epitopes identified along the protein chain. According to this model, epitopes in the LDL receptor binding domain are located on one side of LDL, whereas epitopes located in the N-terminal and Cterminal domains are in close vicinity to each other on the opposite side of LDL [36]. In addition, a prominent protrusion is visible in the images at the pointed end of the particle. A similar knob-like region was apparent in the low resolution model of lipid-free apo-B100 shown in Figure 4. This protrusion most probable represents the non-lipid associated globular N-terminal domain of apo-B100, which shows a high homology to lamprey lipovitellin [58]. Except for the N-terminal domain, little is known about the molecular organisation of the structural motifs, whose amphipathic nature determine lipid association. However, to evaluate lipid-protein interactions physical parameter like interfacial elasticity or molecular dynamics have to be considered. In this context, it was suggested that the hydrophobic β-sheet domains of apo-B100 act as elastic lipid anchors, whereas the amphipathic α-helical domains respond rapidly to changes in surface pressure [59,60]. In any case, it can be assumed that alterations in the adsorption and penetration depth of apo-B100 in the phospholipid monolayer and in the lipid core are accompanied by structural rearrangements of the domains and changes in the orientation of the domains relative to each other. In the course of such elastic motions, intramolecular rearrangements are likely to alter the overall hydrophobicity and surface activity of single protein domains. These modifications not only affect lipid-protein interaction, but are equally important for molecular and cellular recognition of apo-B100.

10 Lipoproteins – Role in Health and Diseases

susceptibility of LDL particles to oxidative modifications and lipid peroxidation might be correlated to temperature [18]. As oxidized LDL play a crucial role in the pathogenesis of atherosclerosis, any contribution to the comprehension of antioxidant efficiency may be of therapeutic potential [2,51], further pointing to the physiological relevance of the lipid core

As already indicated above, the physicochemical state of the core lipids is intimately related to the structure and dynamics of the particle surface, which consists of about 700 phospholipid molecules and one single copy of apo-B100. Apo-B100 is a huge glycoprotein and its polypeptide chain consists of 4536 amino acid residues with an estimated molecular mass of about 550 kDa for the glycosilated form [52,53]. Apo-B100 is a single chain protein with a total contour length of about 70 nm [54] and can be viewed as a highly flexible molecular string composed of single domains [20]. Five consecutive domains were identified based on secondary structure elements representing the main conformational motifs of apo-B100. The single domains are connected by flexible interdomain linker regions, which allow relative movements of domains to each other. The feasibility of such motions was shown in a low resolution model of detergent solubilized apo-B100, which was derived from small angle neutron scattering data [55]. In this model, compact rigid domains are visible being connected by flexible interdomain linkages, which possess a substantial degree of freedom in their spatial orientation. A hypothetical spatial arrangement of the apo-B100 molecule on a spherical LDL particle was created after assigning the secondary structure elements, which were deduced from a secondary structure prediction, to the surface of apo-B100 (Figure 4). Likewise, the averaged surface shape of the 3D-model would allow for variations in the thickness of the apo-B100 molecule by about 1 nm. Such variations are most likely required to compensate for changes in the surface area upon lipid exchange and particle shrinking during endogeneous

**5. Apo-B100 is a flexible string wrapped around the surface of LDL** 

organisation. However, this vital question still remains unanswered.

lipoprotein conversion from very low density lipoprotein (VLDL) to LDL.

correspond to the pentapartite model suggested by [20].

**Figure 4.** Reconstituted low resolution model of lipid-free apo-B100 derived from small angle neutron scattering data. Apo-B100 shows an elongated arch-like morphology indicating single domains and mobile less defined linker regions. A hypothetical model of a spherical LDL particle after superposition of the structural model of apo-B100 is shown (adapted from ref. [55]). Secondary structure modules are assigned to the surface after a secondary structure prediction was performed. The results nicely

#### **6. Apo-B100 containing lipoproteins are very soft and flexible**

LDL particles are formed in the circulation by lipolytic conversion of TG-rich VLDL particles. This enzyme mediated endogenous transformation is accompanied by an extensive shrinking in particle size from about 50-80 nm for VLDL to ~20 nm for LDL. In the course of remodelling, apo-B100 remains bound to its nanocarrier stabilizing the lipid assembly by maintaining structural integrity. To accomplish this, apoB100 has to become more condensed or relaxed depending on the lipid packing density. Likewise, this dynamic process is modulated by the molecular mobility of the surrounding microenvironment. To test for this hypothesis we have recorded temperature dependent molecular motions in VLDL and LDL particles using elastic incoherent neutron scattering [61]. With this technique, motions in the nano- to picosecond time scale can be recorded. The calculated dynamic force constants are a direct measure for the resilience of the particles. The results show that at physiological temperatures VLDL particles are very soft, elastic and mobile as compared to LDL, which is more rigid (see Figure 5). This observation supports the notion that apo-B100 in VLDL is loosely packed at the interface covering a large surface area with

low interfacial surface tension [59]. During particle conversion from VLDL to LDL, however, the relative number of surface molecules increases and a higher molecular packing density leads to a compression of the lipid anchored protein regions and an overall stiffening of the LDL particle [60].

**Figure 5.** Molecular motions in LDL and VLDL. Elastic temperature scans are recorded with elastic incoherent neutron scattering. The mean square thermal fluctuations (<u2>) are shown as function of temperature. The molecular resiliences are derived from the slopes in the curves. It is seen that VLDL has an increased motion at elevated temperatures compared to LDL. Parts of this figure are reproduced, with permission, from ref. [60].

To conclude, the intrinsic conformational flexibility and elasticity of apo-B100 containing lipoprotein particles is most likely critical for specific affinities of lipoproteins to receptors, antibodies or enzymes. Moreover, it would seem that the susceptibility of lipoproteins to oxidative modifications and hence their atherogenicity is influenced by their dynamic nature.

### **7. LDLs are flexible nanotransporters circulating in blood**

In the search for new and improved therapeutics, the field of nanomedicine dealing with functionalized nanoparticles for molecular imaging and therapy is rapidly emerging. Nanoparticles offer new opportunities to transfer active substances directly to the diseased site in the body. By additional surface coatings or functionalizations, the properties of nanoparticles can be tuned to specific needs. Within the last two decades, a variety of artificial nanoparticles have been designed for targeted delivery of drugs or contrast agents. Many of these nanoconstructs are developed for cancer therapy taking advantage of the leaky vasculature of tumours. Apart from tumour targeting, increasing efforts are devoted to the treatment and imaging of atherosclerotic plaques (for a recent review see ref. [62]). Over time, a broad and versatile nanoparticle platform was created in which liposomes and biodegradable polymers have turned out to be the most promising candidates. It is important to mention that several nanomedicine products have already been established on the market and numerous products are successfully applied in clinical trials [63]. However, inherent problems of nanoparticles are biocompatibility and low stability in vivo, since most nanoparticles become rapidly cleared by the reticuloendothelial system. In contrast to artificial systems, lipoproteins are naturally occurring nanoparticles evading recognition by the body´s immune system. Hence, lipoproteins are excellent candidates with attractive properties to be considered as molecular transporters. A great advantage of LDL over other nanoparticles is the fact that LDL particles stay in circulation for several days, and are not cleared immediately by the mononuclear phagocyte system of the liver and spleen. The average lifetime of an LDL particle is 2-3 days and this time span is about three times longer as reported for long-circulating liposomes, currently applied in chemotherapy [64]. It was recognized that certain tumor cells overexpress LDL receptor, however, the targeting specificity is limited as the LDL receptor is ubiquitously expressed throughout the body, most prominent in the liver. However, using apo-B100 as inherent targeting sequence the enhanced circulation times in blood enable drug-loaded LDL particles to bind to specific receptors exposed on the surface of e.g. tumor or atherosclerotic plaque. Once recognized by the receptor, the functionalized LDL particles become internalized, accumulate in the tissue and exert an enhanced effect (reviewed by [65]). The intrinsic targeting properties of LDL to atherosclerotic plaques are already utilized for early diagnosis and detection of atherosclerotic lesions by different imaging modalities (for reviews see refs. [66,67]). However, to modify lipoprotein particles for medical purposes, care has to be taken not to compromise essential biophysical and structural features of LDL with the goal to preserve the biological activity. In general, there are several possibilities to create multifunctionalized lipoprotein particles. Some representative examples are shown in the scheme in Figure 7. One possibility is to load hydrophobic drugs (e.g. chemotherapeutics, antibiotics, vitamins, signal emitting molecules or small nanocrystals) in the lipophilic inner core of LDL. This can be accomplished by different techniques including lyophilisation, solvent evaporation and reconstitution procedures [68,69]. However, LDL particles can not be reconstituted so easily and remote drug/contrast agent loading into native lipoprotein particles is still a tedious approach currently not being standardized. Amphiphilic substances (drugs or marker molecules) or fatty acid modified chelator complexes can be incorporated in the PL monolayer [70,71]. This has successfully been done in numerous biophysical studies and for diagnostic purposes. Finally, the surface of LDL can be modified by protein labeling. This is done by covalent attachment of substances to the lysine and cysteine amino acid residues of apo-B100. Such substances include fluorophores, radionuclides or metal ions for molecular imaging [65]. Alternatively, targeting sequences (e.g. folic acid) can be coupled to apo-B100 with the purpose to reroute LDL to alternate receptors, which, in case of folate, are more specifically expressed in tumor cells [72].

12 Lipoproteins – Role in Health and Diseases

with permission, from ref. [60].

nature.

LDL particle [60].

low interfacial surface tension [59]. During particle conversion from VLDL to LDL, however, the relative number of surface molecules increases and a higher molecular packing density leads to a compression of the lipid anchored protein regions and an overall stiffening of the

**Figure 5.** Molecular motions in LDL and VLDL. Elastic temperature scans are recorded with elastic incoherent neutron scattering. The mean square thermal fluctuations (<u2>) are shown as function of temperature. The molecular resiliences are derived from the slopes in the curves. It is seen that VLDL has an increased motion at elevated temperatures compared to LDL. Parts of this figure are reproduced,

To conclude, the intrinsic conformational flexibility and elasticity of apo-B100 containing lipoprotein particles is most likely critical for specific affinities of lipoproteins to receptors, antibodies or enzymes. Moreover, it would seem that the susceptibility of lipoproteins to oxidative modifications and hence their atherogenicity is influenced by their dynamic

In the search for new and improved therapeutics, the field of nanomedicine dealing with functionalized nanoparticles for molecular imaging and therapy is rapidly emerging. Nanoparticles offer new opportunities to transfer active substances directly to the diseased site in the body. By additional surface coatings or functionalizations, the properties of nanoparticles can be tuned to specific needs. Within the last two decades, a variety of artificial nanoparticles have been designed for targeted delivery of drugs or contrast agents. Many of these nanoconstructs are developed for cancer therapy taking advantage of the

**7. LDLs are flexible nanotransporters circulating in blood** 

**Figure 6.** Scheme giving some examples of how LDL particles can be modified to act as natural endogenous nanoparticles for targeted drug delivery or multifunctional molecular imaging.

To construct lipoprotein mimetic particles, also referred to as lipoprotein related particles, artificial lipoprotein particles have to be reassembled from individual lipid and protein entities. This approach was highly successful for high density lipoproteins using apo-AI mimetic peptide sequences [73]. For LDL, this approach was not pursued yet and will be much more complicated considering the complex dynamic nature of apo-B100.

Over the last few years, a promising nanoparticle platform was established, which exploits the endogenous properties of natural lipoproteins being non-toxic, non-immunogenic and biodegradable. Although this platform still offers vast potential for improvements, first promising results in enhanced multimodal imaging of tumors and atherosclerotic plaques are achieved giving hope that further endeavors to combine diagnostics and personalized therapeutics will also be successful.
